Targeted immune conjugate comprising an antibody to glycophorin A and a M2e peptide

ABSTRACT

Disclosed herein are materials and methods related to vaccines. Materials and methods for delivery of immunogens to the reticuloendothelial system via non-circulating lymphoid cells are provided.

This application is a national stage entry of PCT/US07/71875, filed Jun.22, 2007, which claims benefit of provisional application, U.S. Ser. No.60/816,015, filed Jun. 23, 2006.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 18, 2012, isnamed F5733002.txt and is 3,127 bytes in size.

TECHNICAL FIELD

This invention relates to materials and methods involved inimmunotherapy.

BACKGROUND

Vaccination is one of the most important medical interventions forpreventing disease. The purpose of vaccination is to induce an optimalimmune response that provides preventive or therapeutic benefit to thehost. Vaccines typically contain one or more immunogens (IMGs) that areharmless variants or derivatives of pathogens, which act to stimulatethe immune system to mount defenses against the actual pathogen. Thereare many types of immunogens (IMGs), ranging from attenuated or killedmicroorganisms, microbial extracts, whole proteins, polysaccharides, andpeptides. Some IMGs are very effective inducers of the desired immuneresponse, while others require the co-administration of non-specificimmune stimulants, or adjuvants, or the coupling of the IMG to a carrierprotein or microparticulate substances. Still other IMGs are inherentlypoor at inducing effective immune responses, despite combination withadjuvants and repeated boosts. Many of these inherently weak immunogensare involved in diseases e.g., influenza and cancer, that remain majorcauses of human morbidity and mortality. There is a continuing need foreffective and more potent vaccines, particularly those with the capacityto stimulate a robust immune response against weakly immunogenictargets.

SUMMARY

Disclosed herein are immune conjugates that are useful for inducing orenhancing an immune response against an antigen. Accordingly, disclosedare immune conjugates that include a ligand which binds specifically toa cell surface molecule on a circulating non-lymphoid cell, and animmunogen coupled to the ligand, wherein the immune conjugate, whenadministered to an individual, induces or enhances an immune responseagainst the immunogen. In one embodiment, the immune conjugate includesa ligand which binds specifically to a cell surface molecule on acirculating non-lymphoid cell, wherein the molecule is selected from thegroup consisting of CR2, glycophorin A, band 3, Ter-119, blood groupantigen H, blood group antigen A, blood group antigen B, CD41a, CD14,CD56, CD66d, CD83, CMKLR1, and BDCA-4; and an immunogen coupled to theligand, where the immune conjugate, when administered to an individual,induces or enhances an immune response against the immunogen.

A circulating non-lymphoid cell can be any cell that circulates throughthe body of a mammal in the blood and/or lymph system; and is not a Blymphocyte or a T lymphocyte. The circulating non-lymphoid cell can be,for example, a red blood cell, a platelet, a natural killer cell, amonocyte, a granulocyte or a plasmacytoid dendritic cell. In oneembodiment, the circulating non-lymphoid cell is a red blood cell. Inanother embodiment, the circulating non-lymphoid cell is a platelet. Thecirculating non-lymphoid cell can be derived from any mammal, e.g.,humans, a non-human primates, cattle, horses, pigs, sheep, goats, deer,elk, dogs, cats, mustelids, rabbits, guinea pigs, hamsters, rats, ormice.

The cell-surface molecule can be any molecule that is differentiallyexpressed on circulating non-lymphoid cells relative to the levels ofthe same molecule on a cell type that is not a circulating non-lymphoidcell. The cell-surface molecule can be a polypeptide, a carbohydrate, aphospholipid, or a glycolipid.

In one embodiment, the cell surface molecule can be a molecule on thesurface of a red blood cell, including, for example, complement receptor2 (CR2), glycophorin A (CD235A), band 3 (CD233), TER-119, the ABO bloodgroup antigens, e.g., blood group antigen A, blood group antigen B,blood group antigen H, and phosphatidyl serine. In another embodiment,the cell surface molecule can be a molecule on the surface of aplatelet, including, for example, gpIIb/IIIa (CD41a), CD42d, CD61, CD62P(P-selectin) and CD151. In another embodiment, the cell surface moleculecan be a molecule on the surface of a natural killer (NK) cell,including, for example, CD56 (NCAM, Leu-19, NKH1). In anotherembodiment, the cell surface molecule can be a molecule on the surfaceof a monocyte, including, for example, CD14 (LPS-receptor). In anotherembodiment, the cell surface molecule can be a molecule on the surfaceof a granulocyte, including, for example, CD66d (CGM1, a member of theCEA family). In another embodiment, the cell surface molecule can be amolecule on the surface of a plasmacytoid dendritic cell, including, forexample, CMKLR1 (serpentine chemokine-like receptor 1), BDCA-2 (CD303)and BDCA-4 (CD304).

The ligand can be any molecule capable of binding a specific cellsurface molecule on a circulating non-lymphoid cell. A ligand caninclude, for example, a polypeptide, a carbohydrate, glycolipid orbiomimetic of a polypeptide, carbohydrate or glycolipid, as long as theligand binds specifically to a cell surface molecule that isdifferentially expressed on a circulating non-lymphoid cell.

A ligand can be a polypeptide, provided that it is not C3d, a heat shockprotein, muramyl dipeptide, or muramyl tripeptide.

In one embodiment, a polypeptide ligand can be an antibody. The antibodycan be a monoclonal antibody, a polyclonal antibody, a holoantibody, asingle chain variable fragment immunoglobulin, or a chimeric moleculethat contains the constant region of an immunoglobulin and cell-bindingsequences from a different source grafted in place of the immunoglobulinvariable regions. The antibody can be, for example, an antibody thatrecognizes targets on red blood cells e.g., complement receptor 2 (CR2),glycophorin A (CD235A), band 3 (CD233), TER-119, blood group antigen A,blood group antigen B, and blood group antigen H. The antibody can be,for example, an antibody that recognizes targets on platelets, e.g.gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectin) and CD151. Theantibody can be, for example, an antibody that recognizes targets onmonocytes, e.g., CD14. The antibody can be, for example, an antibodythat recognizes targets on granulocytes, e.g., CD66d. The antibody canbe, for example, an antibody that recognizes targets on NK cells, e.g.,CD56. The antibody can be, for example, an antibody that recognizestargets on plasmacytoid dendritic cells, e.g., CMKLR1, BDCA-2 (CD303)and BDCA-4 (CD304).

A ligand can also be a polypeptide that is not an immunoglobulin,including for example, the erythrocyte-binding antigen 175 (EBA-175) ofPlasmodium falciparum or a fragment of EBA-175 that binds to a red bloodcell, for example, SEQ ID NO:7, a complement fragment that binds to CR2,for example, C4b, C3b, iC3b, C1q, C3d or a peptide derived from thesecomplement fragments, e.g., C3 residues 1201-1214; or a lectin, e.g., aglycoprotein that recognizes blood group antigen A, blood group antigenB or blood group antigen H.

The polypeptide ligand can include post-translational modifications,e.g., biotinylation, glycosylation, acetylation, alkylation,isoprenylation, lipoylation, phosphorylation.

In another embodiment a ligand can also be a carbohydrate or glycolipid,including, for example, bacterial lipopolysaccharide or a fragment ofit, microbial products bound by Toll-like receptors, bacterial diacyland triacyl lipopeptides, lipoteichoic acid or zymosan.

In another embodiment, a ligand can be a nucleic acid, including, forexample, single- and double-stranded viral RNA and CpG DNA.

The immunogen can be any molecule capable of eliciting a functionalimmune response in a mature T or a B lymphocyte or a precursor of a T ora B lymphocyte. Immunogens can include polypeptides, carbohydrates,glycolipids, haptens or biomimetics thereof. The immunogen can be amolecule expressed or released by an infectious agent, including, forexample, viruses, viroids, bacteria, fungi, prions or parasites. Aninfectious agent can include for example, Orthomyxoviridae, e.g.,influenza viruses, including the strain A (H5N1), Rhadboviridae,Hepadnaviridae, e.g., hepatitis B, Picornaviridae, e.g., hepatitis A,Flaviviridae, e.g., hepatitis C; Retroviridae, e.g., humanimmunodeficiency viruses HIV1 and HIV2, Togaviridae, Bunyaviridae, e.g.,hantavirus; Paramyxoviridae, Herpesviridae, Arenaviridae, e.g., lassavirus; Reoviridae; Bacillus anthracis, Clostridium botulinum, Salmonellaenteriditis, Escherichia coli, including E. coli O157:H7, Streptococcuspneumoniae, Staphylococcus aureus, Aspergillus, Stachybotrys, Candida,Cryptosporidium, Toxoplasma, and Plasmodium falciparum.

Immunogens derived from pathogenic organisms can include, for example,influenza A M2 protein, hepatitis B surface antigen, HBV preS1 protein,HIV tat, HIV gp120, anthrax protective antigen, botulinum toxin, andStreptococcus pneumoniae pneumococcal polysaccharides. An influenza M2protein antigen can be the ectodomain peptide M2e, for example SEQ IDNO: 1, or a variant of the ectodomain peptide M2e, for example, SEQ IDNO: 3 or SEQ ID 4. An HBV preS1 protein can include the preS1 proteinpeptide 35-49, for example SEQ ID NO:2.

The immunogen can also be a molecule expressed by a mammal. For example,an immunogen can be a molecule whose expression is correlated with aparticular disease for example, cancer or neurodegenerative disease. Theimmunogen can be a tumor-associated antigen (TAA), including forexample, MART-1, Muc-1, MAGE, RAGE, or CEA. In another embodiment, theimmunogen can be an antigen that is involved in the initiation orprogression of neurodegenerative diseases, e.g. Alzheimer's disease andTransmissible Spongiform Encephalopathies (TSEs), including for example,beta-amyloid, tau protein, alpha synuclein, or a prion-related protein.In another embodiment, the immunogen can be a germ cell antigen,including for example, sperm adhesion molecule 1 (SPAM-1), and humanintra-acrosomal protein. In another embodiment, the immunogen can be anon-toxic variant of a toxic substance, including for example, ricin,botulinum toxins A, B, C, D, E, F and G. nicotine, or a drug of abusesuch as an opiate or opiate derivative.

The ligand and the immunogen are connected by a linker. A linker can beany reagent, molecule or macromolecule that connects the ligand and theimmunogen such that a) the immune complex is stable under physiologicalconditions; b) the connection between the linker and the ligand does notalter the ability of the ligand to bind to its target on the surface ofa circulating non-lymphoid cell; and c) the connection between thelinker and the immunogen does not abolish the capacity of the immunogento induce an effective immune response in a host against an infectiousagent, cell or molecule on which the immunogen is naturally found.

In one embodiment, a linker can be a peptide bond. The ligand and theimmunogen can be a fusion polypeptide comprising one or more amino acidsegments from the ligand and one or more amino acid segments from theimmunogen. The amino acid segments of the ligand can be contiguous withthe amino acid segments of the immunogen or they can be separated byamino acids inserted as a structural spacer. A spacer segment can be oneor more amino acids. The one or more amino acids can include amino acidsthat are the same or that are different. Also encompassed are nucleicacids comprising a nucleotide sequence that encodes the fusion proteins,a vector (e.g., a vector that includes a transcriptional regulatoryelement (TRE) operably linked to the nucleotide sequence) containing thenucleic acid, and a cell (e.g. a prokaryotic cell or a eukaryotic cell)containing the vector.

In another embodiment, the ligand and immunogen can be obtainedseparately, either through chemical synthesis or synthesis in vivo,purified and then linked non-covalently or covalently. The non-covalentlinkage can be a for example, a biotin-avidin (or streptavidin) linkage.The covalent linkage can be through a chemical cross-linking agent, forexample, a homobifunctional cross-linking reagent or aheterobifunctional cross-linking reagent. In another embodiment, theligand and the immunogen can be connected through a linking polymer,including, for example, linear or branched polymers or co-polymers(e.g., polyalkylene, poly(ethylene-lysine), polymethacrylate, polyaminoacids, poly- or oligosaccharides, or dendrimers).

In another embodiment, the ligand and the immunogen can be connectedthrough a microparticle, including, for example, micelles, liposomes,fullerenes, nanotubes, or other colloidal complexes such aslipoproteins. The ligand and the immunogen can be attached to thelinking molecule or microparticle through a non-covalent high affinitylinkage, e.g., avidin-biotin high affinity binding, adsorbed orincorporated into a hydrophobic microparticle by hydrophobic affinity,or covalent chemical cross-linking techniques.

The immune conjugates provided herein can include one or more of thesame ligands or any combination of different ligands. The immuneconjugates can also include one or more of the same immunogens or anycombination of different immunogens.

Also provided are methods and materials for inducing or enhancing animmune response in a mammal, the method comprising administering to themammal an effective amount of a composition comprising an immuneconjugate that includes a ligand which binds specifically to a cellsurface molecule on a circulating non-lymphoid cell, and an immunogencoupled to the ligand, wherein the immune conjugate, when administeredto an individual, induces or enhances an immune response against theimmunogen. In one embodiment, the immune conjugate includes a ligandwhich binds specifically to a cell surface molecule on a circulatingnon-lymphoid cell, wherein the molecule is selected from the groupconsisting of CR2, glycophorin A, band 3, Ter-119, blood group antigenH, blood group antigen A, blood group antigen B, CD41a, CD14, CD56,CD66d, CD83, CMKLR1, and BDCA-4; and an immunogen coupled to the ligand,where the immune conjugate, when administered to an individual, inducesor enhances an immune response against the immunogen.

The mammals can be, for example, humans, non-human primates, horses,cattle, pigs, sheep, deer, elk, goats, dogs, cats, mustelids, rabbits,guinea pigs, hamsters, rats, and mice. The mammal can have, be likely tohave, or be at risk for having, an infectious disease, e.g., a viraldisease, a bacterial disease, a protozooal disease, or a fungal disease.Infectious diseases can include, for example, influenza, HIV-AIDS,hepatitis, botulism, smallpox, viral hemorrhagic fevers,gastrointestinal disease induced by pathogenic forms of E. coli,Salmonella and Shigella, Staphylococcal infection, trypanosomiasis, andmalaria. Alternatively, the mammal can have, be likely to have, or be atrisk for having, a proliferative cell disease, e.g., a cancer such as aneural tissue cancer, melanoma, breast cancer, lung cancer, agastrointestinal cancer, ovarian cancer, testicular cancer, lung cancer,prostate cancer, cervical cancer, bladder cancer, vaginal cancer, livercancer, renal cancer, bone cancer, a hematological cell cancer, or avascular tissue cancer. In another embodiment, the mammal can have, belikely to have, or be at risk for having, a neurodegenerative disease,e.g., Alzheimer's disease or a Transmissible Spongiform Encephalopathy(TSE).

In another embodiment, methods and materials for inducing or enhancingan immune response in a mammal against a germ cell antigen are provided.In a further embodiment, methods and materials for inducing or enhancingan immune response in a mammal against toxic substances, e.g., ricin,botulinum toxin, nicotine and drugs of abuse are provided.

Also provided are compositions featuring immune conjugates that includea ligand which binds specifically to a cell surface molecule on acirculating non-lymphoid cell, and an immunogen coupled to the ligand,wherein the immune conjugate, when administered to an individual,induces or enhances an immune response against the immunogen in apharmaceutically acceptable carrier or excipient. In another embodiment,the composition can include an adjuvant.

Also provided are articles of manufacture that can include immuneconjugates as described herein. An article of manufacture can include,for example, one or more immune conjugates. In addition, an article ofmanufacture further may include, for example, packaging materials,instructions for use, buffers or other control reagents for treating ormonitoring the condition for which prophylaxis or treatment is required.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts the electrophoretic mobility of tripartite immuneconjugates containing biotinylated antibody, streptavidin, and varyingamounts of biotinylated M2e peptide (SEQ ID NO:1).

FIG. 2 depicts the electrophoretic mobility of tripartite immuneconjugates containing biotinylated antibody and a constant ratio ofstreptavidin, and biotinylated M2e peptide (SEQ ID NO:1).

DETAILED DESCRIPTION

The mammalian immune system mounts two different types of responses toimmunogens (IMGs), humoral and cellular. The humoral response, mediatedby B lymphocytes, defends primarily against extracellular pathogensthrough the production of circulating antibodies that mark foreign cellsand molecules for destruction by other specialized cells and proteins.The cellular response, mediated by T lymphocytes, defends predominantlyagainst intracellular pathogens and cancers by directly binding to anddestroying the infected or cancerous cells. Both responses depend uponspecialized cells that internalize through endocytosis, pinocytosis orphagocytosis, and process IMGs; fragments of the IMGs are then presentedto T lymphocytes, which in turn, help to trigger B-lymphoctye responsesagainst the immunogens and/or the T cells to attack the target directly.

The phagocytic cells that function as antigen-presenting cells (APCs)are part of the reticuloendothelial system (RES). The RES is a diffusesystem comprised of circulating and tissue-fixed cells includingmonocytes, macrophages, dendritic cells, Kupffer cells in the liver,Langerhans cells in the skin and microglial cells in the brain Severaltissues and organs, by virtue of their wealth of phagocytic andspecialized endothelial cells, comprise critical locations for theclearance and antigen presentation functions of the RES. These includethe liver, spleen, bone marrow and lymphatic tissues. The RES plays animportant role in clearing potentially harmful materials from the bloodincluding micro-organisms, bacterial endotoxins, immune complexes, tumorcells and senescent and damaged cells of the blood and lymph systems.The cells that cycle through the RES, e.g. red blood cells (orerythrocytes), platelets, natural killer cells, monocytes, granulocytes,and plasmacytoid dendritic cells, serve as carriers of foreignmaterials, which may be stripped off in RES organs. If the load offoreign material on the surface of the circulating cells is very dense,these cells themselves can be targets for phagocytic activity. Inaddition, as circulating cells age, they acquire surface markers thatcause them to be captured by the RES, removed from circulation anddestroyed by phagocytosis, resulting in the degradation of thecirculating cell components and their surface-bound materials. Oncethese cells have been phagocytized, any fragments of macromolecules theyoriginally contained, either endogenously expressed molecules orexogenous molecules that were opsonized to the cell surface canpotentially be presented to T lymphocytes. Thus, molecules bound to redblood cells, platelets, natural killer cells, monocytes, granulocytes,and plasmacytoid dendritic cells can function as immunogens.

Disclosed herein are materials and methods for the specific delivery ofimmunogens to the reticuloendothelial system via circulatingnon-lymphoid cells. In particular, targeted immune conjugates areprovided that specifically bind to circulating non-lymphoid cells, e.g.,red blood cells, platelets, natural killer cells, monocytes,granulocytes, and plasmacytoid dendritic cells, that are cleared throughthe RES. Thus, the circulating non-lymphoid cells act as vehicles forthe specific delivery of IMGs to the RES and thereby to the immunesystem. As used herein, the term “targeting immune conjugate” includes aligand which binds specifically to a cell surface molecule on acirculating non-lymphoid cell coupled to an IMG, where the targetedimmune conjugate, when administered to an individual, induces orenhances an immune response against the IMG. Also provided are methodsof treatment using targeted immune conjugates. Targeted immuneconjugates provide injectable vaccines for efficient immunizationagainst weakly immunogenic IMGs.

II. Compositions

The targeting immune conjugates provided herein have the generalformula:(L)_(y)-X-(IMG)_(z)

-   -   wherein L is a ligand which binds specifically to a cell surface        molecule on a circulating non-lymphoid cell; X is a linker; IMG        is an immunogen, and y and z are integers having a value of one        or greater than one.        Circulating Non-Lymphoid Cells and Targets

As used herein, a circulating non-lymphoid cell can be any cell that a)circulates through the body of a mammal in the blood and/or lymphsystem; and b) is not a B lymphocyte or a T lymphocyte. Thus,circulating non-lymphoid cells include erythrocytes, i.e., red bloodcells; platelets; natural killer cells; monocytes; granulocytes, i.e.,neutrophils, eosinophils, and basophils; and plasmcytoid dendriticcells.

B and T lymphocytes are ultimately derived from hematopoietic stem cellsand perform the principal functions of the immune system. T lymphocytesmature through the thymus and are generally identified by theirexpression of CD3 (which is associated with the T cell receptor) andeither CD4 or CD8. CD8-expressing (or CD8+) T cells are principallyinvolved with direct cell killing, or cytotoxicity. CD4+ T cells areprimarily regulatory cells which stimulate and suppress immune responsesas needed. B lymphocytes are characterized by their expression of CD19or CD20, among other surface markers, and they are responsible forantibody production. B cells are also effective antigen presentingcells.

Erythrocytes, also known as red blood cells, are the most abundant celltype in mammalian blood. They are small disc-shaped, anucleated,biconcave cells whose primary function is to carry oxygen and carbondioxide to and from the tissues. Red blood cells express a distinctivecomplement of cell surface markers, including the human blood groupantigens, glycophorin, band 3 and the Lewis antigens.

Platelets are derived from megakaryocytes, they are centrally involvedin blood clotting, and can be identified by their surface expression ofCD41a (or gpIIb/IIIa). Natural killer cells, also referred to as largegranular lymphocytes, are derived from the bone marrow and do notexpress T-cell antigen receptors (TCR), the pan-T marker CD3 or surfaceimmunoglobulins (Ig) B cell receptor, but typically express the surfacemarkers CD16 (FcγRIII) and CD56. Monocytes are derived from myeloid stemcells and are found primarily in the circulation. They are competentphagocytes. Upon their binding of pathogens and/or stimulation byvarious cytokines, monocytes mature into macrophages, which are evenmore avid phagocytes and producers of many cytokines, degradativeenzymes and other molecules that mediate inflammatory reactions.Macrophages are generally found bound to vascular endothelium or withinvarious tissues. Monocytes (and macrophages) are characterized by thesurface expression of CD14, among other markers.

Granulocytes are also derived from myeloid stem cells and arecharacterized by the presence of abundant granules in their cytoplasm;different classes of granulocytes, e.g., eosinophils, basophils andneutrophils, are distinguished by their ability stain with eosin,basophilic dyes or neither, respectively. Eosinophils are involved indefense against parasitic pathogens and allergens; basophils are alsoinvolved in allergic reactions. Neutrophils are early and aggressivephagocytes at the site of infections and release products that induceinflammatory reactions.

Plasmacytoid dendritic cells (pDC) are distinct from myeloid dendriticcells. Both are found in the circulation and in tissues. pDC are animportant link between the innate and adaptive immune responses, inparticular in mounting anti-viral immune responses. They produceabundant interferons and can be identified by the surface marker BDCA-2(CD303).

The circulating non-lymphoid cells can be derived from any mammal, e.g.,humans, a non-human primates, cattle, horses, pigs, sheep, goats, deer,elk, dogs, cats, mustelids, rabbits, guinea pigs, hamsters, rats, ormice.

Any cell-surface molecule that is differentially expressed oncirculating non-lymphoid cells relative to the levels of the samemolecule on a cell type that is not a circulating non-lymphoid cell is asuitable target for the ligand. The cell-surface molecule can be apolypeptide, a carbohydrate, or a glycolipid. Full-length molecules,epitopes, analogs, mutants, and functional fragments thereof areencompassed by this definition. A “functional fragment” of a molecule isa fragment of the molecule that is smaller (shorter where the moleculeis a polypeptide) than the molecule per se but has at least 10% (e.g.,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 100%or even more) of the ligand-binding activity of the molecule per se.

Typically, a cell-surface molecule on a circulating non-lymphoid cellcan be classified as being differentially expressed if the molecule ispresent at a level that is greater than the average level observed incells that are not circulating non-lymphoid cells if the expressionlevels differ by at least 50% (e.g. 50, 100, 200, 300% or more). Anymethod can be used to determine whether or not a specific gene productis expressed at a level that is greater or less than the average levelof expression observed in control cells. The level of expression of acell surface polypeptide can be measured using any method such asimmuno-based assays (e.g., immunofluorescence, flow cytometry, ELISA),western blotting, or polyacrylamide gel electrophoresis with silverstaining. Levels of particular carbohydrates or lipids can be measuredby immunodetection e.g., ELISA, flow cytometry, or immunostaining usingfluorochrome- or radioisotope-labeled antibodies or lectins. In someembodiments, the level of expression from a particular gene can bemeasured by assessing the level of mRNA expression from the gene. Levelsof mRNA expression can be evaluated using, without limitation, northernblotting, slot blotting, quantitative reverse transcriptase polymerasechain reaction (RT-PCR), or chip hybridization techniques. Such methodscan be used to determine simultaneously the relative expression levelsof multiple mRNAs.

Examples of targets on red blood cells include, without limitation,complement receptor 2 (CR2), glycophorin A (CD235A), band 3 (CD233),TER-119 (Kina et al., Br. J. Hematol. 109: 280-7, 2000), the ABO bloodgroup antigens, e.g., blood group antigen A, blood group antigen B,blood group antigen H, and phosphatidyl serine (Hematology: BasicPrinciples and Practice, R. Hoffman, 2005, 4th ed. New York:Churchill-Livingstone). One useful red blood cell target is TER-119, aregion selectively bound by the antibody, anti-TER-119 and whichcorresponds to the extracellular domain of glycophorin A. Suitableplatelet targets includes gpIIb/IIIa (CD41a), CD42d, CD61, CD62P(P-selectin) and CD151. Natural killer cell targets include, forexample, CD56 (NCAM, Leu-19, HNK1). Monocyte and granulocyte targetsinclude, for example, CD14 (LPS-receptor) and CD66d (CGM1, a member ofthe CEA family), respectively. Plasmacytoid dendritic cell targetsinclude, for example, CMKLR1 (serpentine chemokine-like receptor 1),BDCA-2 (CD303) and BDCA-4 (CD304) (Kuby Immunology, J. Kuby et al.,edt., 2002, 5^(th) edition, W.H. Freeman & Co.).

Ligands

The term “ligand” as used herein refers to a molecule capable of bindinga specific cell surface molecule on a circulating non-lymphoid cell. Aligand can be a polypeptide, a carbohydrate, glycolipid or biomimetic ofa polypeptide, carbohydrate or glycolipid, as long as the ligand bindsspecifically to a cell surface molecule that is differentially expressedon a circulating non-lymphoid cell.

As defined herein, ligands are defined to be “specifically binding”if: 1) they exhibit a threshold level of binding activity, and/or 2)they do not significantly cross-react with related target molecules. Thebinding affinity of a ligand can be readily determined by one ofordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). For example, a liganddisclosed herein can bind to its target with at least 1.5-fold, 2-fold,5-fold, 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold orgreater affinity for the target than for a closely related or unrelatedpolypeptide. A ligand can bind its target with high affinity (10⁻⁴M orless, 10⁻⁷M or less, 10⁻⁹M or less, or with subnanomolar affinity (0.9,0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM or even less). Ligands canalso be described or specified in terms of their binding affinity to atarget, for example, binding affinities include those with a Kd lessthan 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M,10⁻⁵M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸ M, 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M, or less.

In some embodiments, the ligands disclosed herein do not bind to knownrelated molecules. In other embodiments, the ligands disclosed hereincan bind to orthologs, homologs, paralogs or variants, or combinationsand subcombinations thereof, of their targets.

As defined herein, a ligand that specifically binds to circulatingnon-lymphoid cells is a ligand the binds to circulating non-lymphoidcells and does not significantly bind to lymphoid cells. For example, animmune conjugate that includes a ligand that specifically binds to redblood cells will bind to red blood cells and not significantly bind toother circulating cells, e.g., lymphocytes, platelets, natural killercells, monocytes, granulocytes or dendritic cells. In another example,an immune conjugate that includes a ligand that specifically binds toplatelets will bind to platelets and not significantly bind to othercirculating cells, e.g., lymphoid cells, red blood cells, natural killercells, monocytes, granulocytes or dendritic cells.

Ligands may be screened against known related target polypeptides toisolate a ligand that specifically binds the target. For example, aligand specific to a target will flow through an affinity chromatographycolumn comprising other closely related target molecules adhered toinsoluble matrix under appropriate buffer conditions. Such screeningallows isolation of ligands non-crossreactive to closely related targets(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; Current Protocols in Immunology, Cooliganet al. (eds.), National Institutes of Health, John Wiley and Sons, Inc.,1995). Screening and isolation of specific antibodies is well known inthe art (see, Fundamental Immunology, W. Paul (ed.), Raven Press, 1993;Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies:Principles and Practice, Goding, J. W. (ed.), Academic Press Ltd., 1996;Benjamin et al., Am. Rev. Immunol. 2: 67-101, 1984). Representativeexamples of such assays include: concurrent immunoelectrophoresis,radioimmunoassay (RIA), radioimmunoprecipitation, flow cytometry, FACS,enzyme-linked immunosorbent assay (ELISA), dot blot or western blotassay, inhibition or competition assay, and sandwich assay.

A ligand can be a polypeptide, provided that it is not C3d, a heat shockprotein, muramyl dipeptide, or muramyl tripeptide. The term“polypeptide” as used herein refers to a compound of two or more subunitamino acids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification, e.g., phosphorylation or glycosylation.The subunits may be linked by peptide bonds or other bonds such as, forexample, dicysteine, ester or ether bonds. The term “amino acid” refersto natural and/or unnatural or synthetic amino acids, including D/Loptical isomers. Full-length proteins, analogs, mutants, and fragmentsthereof are encompassed by this definition.

The amino acid sequence of the ligands disclosed herein can be identicalto the wild-type sequences of appropriate components. Alternatively, anyof the components can contain mutations such as deletions, additions, orsubstitutions. All that is required is that the variant ligand have atleast 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,100%, or even more) of the ability of the ligand containing onlywild-type sequences to specifically bind the target on the circulatingnon-lymphoid cell. Substitutions will preferably be conservativesubstitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine.

Any method can be used to make a polypeptide including, for example,expression by prokaryotic systems, expression by eukaryotic systems, andchemical synthesis techniques. Any method can be used to purify apolypeptide including, without limitation, fractionation,centrifugation, and chromatography, e.g., gel filtration, ion exchangechromatography, reverse-phase HPLC and immunoaffinity purification.

A suitable polypeptide ligand can be an antibody. In one embodiment, theantibody can be a monoclonal antibody, i.e., homogeneous antibodies ofidentical antigenic specificity produced by a single clone ofantibody-producing cells. In another embodiment, the antibody can be apolyclonal antibody, i.e., heterogenous antibodies that can recognizedifferent epitopes on the same antigen and that are produced by morethan one clone of antibody producing cells.

The antibody can include any class of immunoglobulin, e.g. IgG, IgA,IgM; and can be derived from any species e.g., humans, mice, rats, orcan be a humanized version of a non-human antibody. An antibody caninclude, without limitations, a holoantibody, i.e., an antibody thatincludes one or more of an immunoglobulin monomer units of two heavy andtwo light chains, a single chain variable fragment immunoglobulin, or achimeric molecule that contains the constant region of an immunoglobulinand cell-binding sequences from a different source grafted in place ofthe immunoglobulin variable regions. The cell-binding regions can befrom a different antibody, a lectin, a cytokine, a microbial proteinfragment or any other molecule that binds the target cell receptormolecule with specificity.

Methods for producing antibodies are well know to those in the art;Antibodies can be purified by chromatographic methods known to those ofskill in the art, including ion exchange and gel filtrationchromatography (for example, Caine et al., Protein Expr. Purif. (1996)8(2):159-166). Alternatively or in addition, antibodies can be purchasedfrom commercial sources, for example, Invitrogen (Carlsbad, Calif.); MPBiomedicals (Solon, Ohio); Nventa Biopharmaceuticals (San Diego, Calif.)(formerly Stressgen); Serologicals Corp. (Norcross, Ga.).

The antibody can be, for example, an antibody that recognizes targets onred blood cells, including for example, without limitation, glycophorinA (CD235A), band 3 (CD233), TER-119, blood group antigen A, blood groupantigen B, and blood group antigen H. One useful antibody isanti-TER-119, which specifically binds to TER-119, a regioncorresponding to the extracellular domain of glycophorin A. Othersuitable antibodies include, without limitation, antibodies thatrecognize targets on platelets, e.g., gpIIb/IIIa (CD41a), CD42d, CD61,CD62P (P-selectin) and CD151; monocytes, e.g., CD14; NK cells, e.g.,CD56; granulocytes, e.g., CD66d; and plasmacytoid dendritic cells, e.g.,CMKLR1, BDCA-2 (CD303) and BDCA-4 (CD304).

A ligand can also be a polypeptide that is not an immunoglobulin. Onenon-immunoglobulin type ligand can be the erythrocyte-binding antigen175 (EBA-175) of Plasmodium falciparum, which specifically binds the redblood cell surface protein band 3, or a fragment of EBA-175 that bindsto a red blood cell, for example EBA-175 peptide 1085-96, SEQ ID NO:7.Another polypeptide ligand can also be a complement fragment that bindsto CR2, for example, C4b, C3b, iC3b, C1q or a peptide derived from thecomplement fragments, e.g., C3 residues 1201-1214 (Tsokos et al.,Journal of Immunol. 144: 1640-45, 1990) or residues 727-768 (Becherer,Biochemistry 31(6):1787-94, 1992). A polypeptide ligand can also be alectin, e.g. a glycoprotein that recognizes blood group antigen A, bloodgroup antigen B or blood group antigen H.

A ligand can also be a peptidomimetic, a small protein-like chaincontaining non-peptidic structural elements that is capable of mimickingor antagonizing the biological action(s) of a natural parent peptide.Peptidomimetic compounds are synthetic, non-peptide compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to bind the ligand in a manner qualitatively identicalto that of the parent peptide from which the peptiomimetic was derived.Peptidomimetic compounds can have additional characteristics thatenhance their therapeutic utility, such as increased prolongedbiological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

Any peptidomimetic that binds specifically and selectively to a cellsurface molecule that is differentially expressed on a circulatingnon-lymphoid cell can be used. Examples of useful peptidomimeticsinclude those that mimic the ability of antibodies that recognize, forexample, complement receptor 1 (CR1), complement receptor 2 (CR2),glycophorin A (CD235A), band 3 (CD233), TER-119, blood group antigen A,blood group antigen B, and blood group antigen H, gpIIb/IIIa (CD41a),CD42d, CD61, CD62P (P-selectin), CD151, CD14, CD56, CD66d, CMKLR1 andBDCA-2.

The polypeptide can include post-translational modifications, i.e.,chemical modification of the polypeptide after its synthesis. Chemicalmodifications can be naturally occurring modifications made in vivofollowing translation of the mRNA encoding the polypeptide or syntheticmodifications made in vitro. A polypeptide can include one or morepost-translational modifications, in any combination of naturallyoccurring, i.e., in vivo, and synthetic modifications made in vitro.Examples of post-translational modifications include, but are notlimited to, biotinylation, e.g., acylation of lysine or other reactiveamino acid residues with a biotin molecule; glycosylation, e.g.,addition of a glycosyl group to either asparagines, hydroxylysine,serine or threonine residues to generate a glycoprotein or glycopeptide;acetylation, e.g. the addition of an acetyl group, typically at theN-terminus of a polypeptide; alkylation, e.g., the addition of an alkylgroup; isoprenylation, e.g., the addition of an isoprenoid group;lipoylation, e.g. attachment of a lipoate moeity; phosphorylation, e.g.addition of a phosphate group to serine, tyrosine, threonine orhistidine.

A highly suitable post-translational modification can be biotinylation.Biotin, also known as vitamin H or B7 is a water-soluble B-complexvitamin that binds avidin or streptavidin with very high affinity(10⁻¹⁵M). Both egg avidin and bacterial streptavidin have 4biotin-binding sites and thus can serve to couple several biotinylatedligands or to couple biotinylated ligand(s) to other biotinylatedmolecules (e.g., IMGs). Polypeptides can be covalently linked to one ormore biotin molecules through primary amines, e.g. lysine andN-terminus), carboxyl groups found on aspartic- and glutamic-acidresidues and at the C-terminus, sulfhydryl groups, or carbohydratemodifications on glycoproteins. Methods for derivatizing polypeptideswith biotin are well known in the art and there are many commercialsources for such reagents (e.g. Pierce, Sigma-Aldrich). Any method ofbiotinylation can be used that preserves the ability of the ligand tobind to its target on the non-circulating lymphoid cell. For example,desirable biotinlyation methods would modify residues in the Fc portionof the antibody without compromising the immunoreactivity of theantibody. Polypeptides derivatized with biotin can then be linked toimmunogens through an avidin or streptavidin molecule.

A post-translational modification can be glycosylation, i.e., theaddition of saccharides. Glycosylation is typically classified based onthe amino acid through which the saccharide linkage occurs and caninclude: N-linked glycosylation to the amide nitrogen of asparaginesside chains, O-linked glycosylation to the hydroxyloxygen of serine andthreonine side chains, and C-mannosylation.

A ligand can also be a carbohydrate or glycolipid. Examples ofcarbohydrate and glycolipid ligands, include, without limitation,bacterial lipopolysaccharide (LPS) or a fragment of it, microbialproducts bound by Toll-like receptors (TLRs), bacterial diacyl andtriacyl lipopeptides and lipoteichoic acid from bacteria, and zymosanfrom yeast cell walls.

A ligand can also be a nucleic acid. Examples of nucleic acids include,without limitation, single- and double-stranded RNA from viruses, andCpG DNA from bacteria or viruses.

Immunogens

As defined herein, an immunogen is any molecule capable of eliciting afunctional immune response (e.g., a cytotoxic or helper T cell responseor an antibody producing response) in a T or a B cell.

As used herein, an “effector T lymphocyte” is a T lymphocyte havingimmunological activity. Such immunological activity can be, withoutlimitation, cytotoxic activity, helper activity, suppressive activity,immune-deviating activity, inflammatory activity, or pro-inflammatoryactivity. As used herein, an “effector T lymphocyte precursor cell” is aT lymphocyte that, subsequent to activation, has any of the aboveimmunological activities. Activation can occur, without limitation, byrecognition of a complex of the relevant immunogenic peptide epitope andthe major histocompatibility complex (MHC) molecule by the T cellreceptor (TCR) on the effector T lymphocyte or by a non-specificstimulus, e.g., a T cell mitogen such as concanavalin A. Thus, aneffector T lymphocyte cell can be a “virgin” T lymphocyte that has neverpreviously been activated or a “memory” T lymphocyte that has previouslybeen activated or the progeny of such a memory T lymphocyte. As usedherein, a “cytotoxic T lymphocyte” (CTL) is a T lymphocyte that can killa target cell expressing on its surface a peptide epitope-MHC molecularcomplex for which the TCR of the CTL is specific.

As used herein, a “CTL cell” is a T lymphocyte that can, subsequent toactivation, kill a target cell expressing on its surface a peptideepitope-MHC molecular complex for which the TCR of the CTL is specific.Activation can be, without limitation, by recognition of the relevantpeptide epitope-MHC molecular complex by a TCR on the CTL or by anon-specific stimulus, e.g., a T cell mitogen such as concanavalin A.Thus, a CTL cell can be a “virgin” T lymphocyte that has neverpreviously been activated or a “memory” T lymphocyte that has previouslybeen activated or the progeny of such a memory T lymphocyte.

As used herein, a “helper T lymphocyte” (Th) is a T lymphocyte thatprovides helper or regulatory activity in an immune response. Such animmune response can be, for example, an antibody-producing response of aB lymphocyte, a response of a CTL precursor cell, or an inflammatory orpro-inflammatory response of a variety of leukocyte types. As usedherein, a “Th cell” is a T lymphocyte that, subsequent to activation,provides helper activity in an immune response such as those listedabove for Th. Activation can be as indicated above for CTL cells.Furthermore, a Th cell can be a “virgin” T lymphocyte that has neverpreviously been activated or a “memory” T lymphocyte that has previouslybeen activated or the progeny of such a memory T lymphocyte.

As used herein, a B cell is a B lymphocyte that, subsequent toactivation, can produce antibody molecules. Activation of a B cell canbe, without limitation, by recognition of an antigen by an antigenspecific immunoglobulin receptor on the B cell surface or by anon-specific stimulus, e.g., a B cell mitogen such as lipopolysaccharideor pokeweed mitogen. Thus, a B cell can be a “virgin” B lymphocyte thathas never previously been activated or a “memory” B lymphocyte that haspreviously been activated or the progeny of such a B lymphocyte.

As used herein, “antigenic” means capable of being recognized by aneffector lymphocyte or an antibody molecule. Thus a substance isantigenic if it is recognized by an antigen specific receptor on, forexample, a CTL, a Th, or a B lymphocyte producing antibody molecules orby an antibody molecule physically unassociated with a B lymphocyte.

An immunogen can be a polypeptide, carbohydrate, glycolipid, hapten orbiomimetic thereof. A polypeptide immunogen, as defined above, caninclude without limitation, a compound of two or more subunit aminoacids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification, e.g. phosphorylation or glycosylation.The subunits may be linked by peptide bonds or other bonds such as, forexample, ester or ether bonds. The term “amino acid” refers to naturaland/or unnatural or synthetic amino acids, including D/L opticalisomers. Full-length proteins, analogs, mutants, and fragments thereofare encompassed by this definition.

The amino acid sequence of the immunogens disclosed herein can beidentical to the wild-type sequences of appropriate components.Alternatively, any of the components can contain mutations such asdeletions, additions, or substitutions. All that is required is that thevariant ligand have at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of theimmunogen containing only wild-type sequences to induce an immuneresponse against the naturally occurring wild-type immunogen.Substitutions will preferably be conservative substitutions.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine, glutamine, serine andthreonine; lysine, histidine and arginine; and phenylalanine andtyrosine.

A polypeptide immunogen can include any peptide epitopes of a variety oflengths, for example, 7-50 (e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38,40, 45, or 50) amino acid residues long. A polypeptide immunogen caninclude one or more epitopes, for example, 1, 2, 4, 6, 10, 20, 30, 50 ormore.

Polypeptide immunogens can include one or more post-transcriptionalmodifications as described above in the Ligands section. An immunogencan be, without limitation, biotinylated, glycosylated, acetylated,alkylated, isoprenylated, lipoylated, or phosphorylated.

An immunogen can also be a molecule that is not a protein, e.g. acarbohydrate or glycolipid. Examples of carbohydrate and glycolipidimmunogens, include, without limitation, bacterial lipopolysaccharide(LPS) or a fragment of it, microbial products bound by various Toll-likereceptors (TLRs), bacterial diacyl and triacyl lipopeptides andlipoteichoic acid from bacteria, and zymosan from yeast cell walls.

The immunogen can be present in a killed or attenuated organism, in acrude cellular extract, a cell lysate or partially or substantiallypure. The term “substantially pure” with respect to anaturally-occurring immunogen refers to an immunogen that has beenseparated from cellular components by which it is naturally accompanied,such that it is at least 60% (e.g., 70%, 80%, 90%, 95%, or 99%), byweight, free from naturally-occurring organic molecules with which it isnaturally associated. Methods for purifying immunogens are known tothose in the art. For example, in general, a substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

The immunogen can be a molecule expressed or released by any of a widerange of infectious agents, including, without limitation, viruses,viroids, bacteria, fungi, prions or parasites.

For example, viral pathogens can include, without limitation, influenzaviruses, including the strain A (H1N5), hepatitis viruses (e.g,Hepatitis A, B, C and D), Arenaviruses, Bunyaviruses, Flaviviruses,Filoviruses, Alphaviruses, (e.g., Venezuelan equine encephalitis,eastern equine encephalitis, western equine encephalitis), Hantaviruses,human immunodeficiency viruses HIV1 and HIV2, feline immunodeficiencyvirus, simian immunodeficiency virus, measles virus, rabies virus,rotaviruses, papilloma virus, respiratory syncytial virus, Variola, andviral encephalitides, (e.g., West Nile Virus, LaCrosse, Californiaencephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus, KyasanurForest Virus). Bacterial pathogens can include, but are not limited to,Bacillus anthracis, Yersinia pestis, Yersinia enterocolitica,Clostridium botulinum, Clostridium perfringens Francisella tularensis,Brucella species, Salmonella spp., including Salmonella enteriditis,Escherichia coli including E. coli O157:H7, Streptococcus pneumoniae,Staphylococcus aureus, Burkholderia mallei, Burkholderia pseudomallei,Chlamydia spp., Coxiella burnetii, Rickettsia prowazekii, Vibrio spp.,Shigella spp. Listeria monocytogenes, Mycobacteria tuberculosis, M.leprae, Borrelia burgdorferi, Actinobacillus pleuropneumoniae,Helicobacter pylori, Neisseria meningitidis, Bordetella pertussis,Porphyromonas gingivalis, and Campylobacter jejuni.

Fungal pathogens can include, without limitation, members of the generaAspergillus, Penecillium, Stachybotrys, Trichoderma, mycoplasma,Histoplasma capsulatum, Cryptococcus neoformans, Chlamydia trachomatis,and Candida albicans.

Pathogenic protozoa can include, for example, members of the generaCryptosporidium, e.g., Cryptosporidium parvum, Giardia lamblia,Microsporidia and Toxoplasma, e.g., Toxoplasma brucei, Toxoplasmagondii, Entamoeba histolytica, Plasmodium falciparum, Leishmania majorand Cyclospora cayatanensis.

Examples of useful immunogens derived from pathogenic organisms include,for example, but are not limited to, influenza A M2 protein, hepatitis Bsurface antigen, HBV preS1 protein, HIV tat, HIV gp120, anthraxprotective antigen, and botulinum toxin. An influenza M2 protein antigencan be the ectodomain peptide M2e, for example, SLLTEVETPIRNEWGCRCNDSSD(SEQ ID NO: 1), or a variant of the ectodomain peptide M2e, for example,SEQ ID NO: 3 or SEQ ID 4. An HBV preS1 protein can include the preS1protein peptide 35-49, e.g., FGANSNNPDWDFNPNKDHWPEANQVGA (SEQ ID NO:2).Examples of useful non-peptidic immunogens include the pneumococcalpolysaccharides from Streptococcus pneumoniae.

The immunogen can also be a molecule expressed by a mammal. For example,an immunogen can be a molecule whose expression is correlated with aparticular disease state, for example, cancer or neurodegenerativedisease.

Thus, the immunogen can be a tumor-associated antigen (TAA). As usedherein, a TAA is a molecule (e.g., a polypeptide, carbohydrate or lipid)that is expressed by a tumor cell and either (a) differs qualitativelyfrom its counterpart expressed in normal cells, or (b) is expressed at ahigher level in tumor cells than in normal cells. Thus, a TAA can differ(e.g., by one or more amino acid residues where the molecule is aprotein) from, or it can be identical to, its counterpart expressed innormal cells. Preferably it is not expressed by normal cells.Alternatively, it is expressed at a level at least two-fold higher(e.g., a two-fold, three-fold, five-fold, ten-fold, 20-fold, 40-fold,100-fold, 500-fold, 1.000-fold, 5.000-fold, or 15.000-fold higher) in atumor cell than in the tumor cell's normal counterpart. Examples ofrelevant cancers include, without limitation, hematological cancers suchas leukemias and lymphomas, neurological tumors such as astrocytomas orglioblastomas, melanoma, breast cancer, lung cancer, head and neckcancer, gastrointestinal tumors such as gastric or colon cancer, livercancer, pancreatic cancer, genitourinary tumors such ovarian cancer,vaginal cancer, bladder cancer, testicular cancer, prostate cancer orpenile cancer, bone tumors, and vascular tumors. Relevant TAAs include,without limitation, carcinoembryonic antigen (CEA), RAGE, MART (melanomaantigen), MAGE (melanoma antigen) 1-4, 6 and 12, MUC (mucin) (e.g.,MUC-1, MUC-2, etc.), tyrosinase, Pmel 17 (gp100), GnT-V intron Vsequence (N-acetylglucoaminyltransferase V intron V sequence), Prostatecancer psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanomaubiquitous mutated gene product), GAGE (melanoma antigen)₁, BAGE(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virusnuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53,lung resistance protein (LRP) Bc1-2, prostate specific antigen (PSA),and Ki-67.

Other immunogens that can be included in the immune conjugates disclosedherein are those derived from antigens that are involved in theinitiation or progression of neurodegenerative diseases, e.g.Alzheimer's disease and Transmissible Spongiform Encephalopathies(TSEs), e.g., human prion diseases such as Creutzfeld-Jacob disease(CJD), variant CJD (“mad cow disease”), Gerstmann-Straussler-Scheinkersyndrome (GSS); Fatal familial Insomnia (FFI); animal prion diseasessuch as Scrapic in sheep; bovine spongiform encephalopathy (BSE) incows; transmissible mink encephalopathy (TME) in mink; chronic wastingdisease (CWD) in elk and deer.

As used herein, a “neurodegenerative antigen” is a molecule (e.g., apolypeptide, carbohydrate or lipid) that is expressed by a neuronal cellin an individual with a neurodegenerative disease and either (a) differsqualitatively from its counterpart expressed in cells from an individualwho does not have the neurodegenerative disease, e.g., the moleculeappears in abnormal locations within the body or is associated withother molecules not normally found with the antigen in healthyindividuals who do not have the neurodegenerative disease, or (b) isexpressed at a higher level in cells from an individual who does nothave the neurodegenerative disease. Thus, a neurodegenerative antigencan differ (e.g., by one or more amino acid residues where the moleculeis a protein) from, or it can be identical to, its counterpart expressedin normal cells. It is preferably not expressed by normal cells.Alternatively, it is expressed at a level at least two-fold higher(e.g., a two-fold, three-fold, five-fold, ten-fold, 20-fold, 40-fold,100-fold, 500-fold, 1,000-fold, 5,000-fold, or 15,000-fold higher) in atumor cell than in the tumor cell's normal counterpart.

Examples of neurodegenerative antigens found in Alzheimer's diseaseinclude beta-amyloid, tau protein, alpha synuclein. Otherneurodegenerative disease antigens can be derived from prions. Asdefined herein, a prion is small proteinaceous infectious particle thatresists inactivation by procedures that modify nucleic acids. Prions areencoded by the prion-related protein gene (PrP). Mutant forms of the PrPprotein aggregate as prions which can lead to fatal neurodegenerativedisease. Thus, an immunogen can be a PrP polypeptide.

Germ cell immunogens can be useful in the generation of immune responsesthat block the function of germ cells, thereby interfering withconception. Germ cell antigens can include antigens on sperm cells.Examples include, without limitation sperm adhesion molecule 1 (SPAM-1),and human intra-acrosomal protein

An immunogen can also be a non-toxic variant of a toxic substance (a“toxoid”) that can be used to stimulate an immune response against theharmful form of the toxin. A toxoid can be, without limitation, a toxinthat has been rendered less toxic or completely non-toxic throughtreatment with high temperature, aggregation, chemical reaction (e.g.,formalin fixation), coupling to a carrier molecule, or molecularalteration (e.g., deletion, augmentation or substitution). A toxoid canbe thus derived from a toxin such as, for example, ricin, anthrax orbotulinum toxin types A, B, C, D, E, F or G.

An immunogen can also be a substance of abuse such as nicotine, or anopiate or opiate derivative. Such an immunogen can induce antibodiescapable of binding and neutralizing the corresponding substance ofabuse.

Forms of Immune Conjugates

The ligand and the immunogen are connected by a linker. A linker can beany reagent, molecule or macromolecule that connects the ligand and theimmunogen such that a) the immune complex is stable under physiologicalconditions; b) the connection between the linker and the ligand does notalter the ability of the ligand to bind to its target on the surface ofa circulating non-lymphoid cell; and c) the connection between thelinker and the immunogen does not substantially affect the capacity ofthe immunogen to induce an effective immune response in a host againstan infectious agent, cell or molecule on which the immunogen isnaturally found.

Fusion proteins. A linker can be a peptide bond. That is, the ligand andthe immunogen can be a fusion polypeptide comprising one or more aminoacid segments from the ligand and one or more amino acid segments fromthe immunogen. The term “amino acid segment” as used herein refers to acontiguous stretch of amino acids within a polypeptide. For example, theamino acid residues 30 to 40 within a 100 amino acid polypeptide wouldbe considered an amino acid segment. An amino acid segment can be alength greater than eight amino acid residues (e.g. greater than aboutnine, ten, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 500, 1000, or moreamino acid residues). In some embodiments, an amino acid segment canhave a length less than 1000 amino acid residues (e.g., less than 500,less than 400, less than 350, less than 300, less than 200, or less than100 amino acid residues). In other embodiments, an amino acid segmentcan have a length from about 20 to about 200 amino acid residues (e.g.about 30 to about 180 amino acid residues, or about 40 to about 150amino acid residues).

The amino acid segments of the ligand can be contiguous with the aminoacid segments of the immunogen or they can be separated by amino acidsinserted as a structural spacer. A spacer segment can be one or moreamino acids. The one or more amino acids can include amino acids thatare the same or that are different. For example, a spacer can be arepeating series of a neutral amino acid (e.g., glycine, alanine,valine, isoleucine or lencine) ranging in number from 1 to 10 or more(e.g., 1, 2, 3, 4, 5, 6, 7, 9, 10 or more). Another example of a spacerconfiguration can be a series of interspersed amino acids that may beneutral (e.g. [glycine-alanine-glycine-alanine-glycine-alanine] (SEQ IDNO: 8) or [glycine-glycine-glycine-valine-valine-valine[ (SEQ ID NO: 9)or charged amino acids (e.g.,glutamate-glutamate-glutamate-arginine-arginine-arginine](SEQ ID NO: 10) or[aspartate-lysine-aspartate-lysine -aspartate-tysine]) (SEQ ID NO: 11)or amino acids with other functional groups (e.g.,[proline-proline-proline-serine-serine-serine](SEQ ID NO: 12)orityrosine-glutamine-cysteine-methionine-tryptophan]) (SEQ NO: 13)ranging in number from 1 to 10 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,20 or more). In another embodiment, a spacer configuration can be asequence of amino acids derived from a naturally occurring protein suchas the hinge region joining the heavy chain CH1 and CH2 domains ofimmunoglobulin G.

A fusion protein can be produced in vitro by continuous peptidesynthesis according to standard chemical methods know to those in theart. Synthetic polypeptides can also be purchased from commercialsources.

A fusion protein can also be produced by recombinant DNA techniques.Nucleic acid segments encoding the ligand can be operably linked in thesame open reading frame to nucleic acid sequences encoding the immunogenin a vector that includes the requisite regulatory elements, e.g.,promoter sequences, transcription initiation sequences, and enhancersequences, for expression in prokaryotic or eukaryotic cells. Methodswell known to those skilled in the art can be used to constructexpression vectors containing relevant coding sequences and appropriatetranscriptional/translational control signals. Alternatively, suitablevector systems can be purchased from commercial sources.

Nucleic acid segments encoding ligands and immunogens are readilyavailable in the public domain. Examples of nucleic acid segmentsencoding ligands include, without limitation, the erythrocyte(glycophorin A)-binding antigen of Plasmodium falciparum EBA-175(Bharara et al., Mol. Biochem. Parasitol. 138: 123-9, 2004), or mouseanti-human glycophorin A monoclonal antibody heavy chain (GenBankaccession # AAZ67132) and corresponding light chain (Genbank accession #AAA21366)). Examples of nucleic acid segments encoding immunogensinclude, without limitation, Hepatitis virus C polyprotein (Genbankpublic gi number 2654998); influenza virus A conserved M2 proteinectodomain peptide M2e (Genbank public gi number gi 78210829; HBV preS1protein (Genbank public gi number gi 92111469).

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein, and refer to both RNA and DNA, including cDNA, genomic DNA,synthetic DNA, and DNA (or RNA) containing nucleic acid analogs.Polynucleotides can have any three-dimensional structure. A nucleic acidcan be double-stranded or single-stranded (i.e., a sense strand or anantisense strand). Non-limiting examples of polynucleotides includegenes, gene fragments, exons, introns, messenger RNA (mRNA), transferRNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers, as well as nucleic acid analogs. The nucleic acid moleculescan be synthesized (for example, by phosphoramidite based synthesis) orobtained from a biological cell, such as the cell of a mammal. Thenucleic acids can be those of mammal, e.g., humans, a non-humanprimates, cattle, horses, pigs, sheep, goats, deer, elk, dogs, cats,mustelids, rabbits, guinea pigs, hamsters, rats, or mice.

An “isolated” nucleic acid can be, for example, a naturally-occurringDNA molecule, provided one of the nucleic acid sequences normally foundimmediately flanking that DNA molecule in a naturally-occurring genomeis removed or absent. Thus, an isolated nucleic acid includes, withoutlimitation, a DNA molecule that exists as a separate molecule,independent of other sequences (e.g., a chemically synthesized nucleicacid, or a cDNA or genomic DNA fragment produced by the polymerase chainreaction (PCR) or restriction endonuclease treatment). An isolatednucleic acid also refers to a DNA molecule that is incorporated into avector, an autonomously replicating plasmid, a virus, or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

Isolated nucleic acid molecules can be produced by standard techniques.For example, polymerase chain reaction (PCR) techniques can be used toobtain an isolated nucleic acid containing a nucleotide sequencedescribed herein. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Various PCR methods are described, for example, in PCRPrimer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold SpringHarbor Laboratory Press, 1995. Generally, sequence information from theends of the region of interest or beyond is employed to designoligonucleotide primers that are identical or similar in sequence toopposite strands of the template to be amplified. Various PCR strategiesalso are available by which site-specific nucleotide sequencemodifications can be introduced into a template nucleic acid. Isolatednucleic acids also can be chemically synthesized, either as a singlenucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector. Isolated nucleic acids disclosed herein also canbe obtained by mutagenesis of, e.g. a naturally occurring DNA.

As used herein, the term “percent sequence identity” refers to thedegree of identity between any given query sequence and a subjectsequence. A subject sequence typically has a length that is more than 80percent, e.g., more than 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105,110, 115, or 120 percent, of the length of the query sequence. A querynucleic acid or amino acid sequence can be aligned to one or moresubject nucleic acid or amino acid sequences using the computer programClustalW (version 1.83, default parameters), which allows alignments ofnucleic acid or protein sequences to be carried out across their entirelength (global alignment). Chema et al., Nucleic Acids Res.,31(13):3497-500 (2003). ClustalW can be run, for example, at the BaylorCollege of Medicine Search Launcher site(searchlauncher.bcm.tmc.edu/multi-Align/multi-align.html) and at theEuropean Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

The term “exogenous” with respect to a nucleic acid indicates that thenucleic acid is part of a recombinant nucleic acid construct, or is notin its natural environment. For example, an exogenous nucleic acid canbe a sequence from one species introduced into another species, i.e., aheterologous nucleic acid. Typically, such an exogenous nucleic acid isintroduced into the other species via a recombinant nucleic acidconstruct. An exogenous nucleic acid can also be a sequence that isnative to an organism and that has been reintroduced into cells of thatorganism. An exogenous nucleic acid that includes a native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid.

A “vector” is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Suitablevector backbones include, for example, those routinely used in the artsuch as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs.The term “vector” includes cloning and expression vectors, as well asviral vectors and integrating vectors. An “expression vector” is avector that includes a regulatory region. Suitable expression vectorsinclude, without limitation, plasmids and viral vectors derived from,for example, bacteriophage, baculoviruses, and retroviruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

Vectors typically contain one or more regulatory regions. The term“regulatory region” refers to nucleotide sequences that influencetranscription or translation initiation and rate, and stability and/ormobility of a transcription or translation product. Regulatory regionsinclude, without limitation, promoter sequences, enhancer sequences,response elements, protein recognition sites, inducible elements,protein binding sequences, 5′ and 3′ untranslated regions (UTRs),transcriptional start sites, termination sequences, polyadenylationsequences, and introns.

As used herein, the term “operably linked” refers to positioning of aregulatory region and a sequence to be transcribed in a nucleic acid soas to influence transcription or translation of such a sequence. Forexample, to bring a coding sequence under the control of a promoter, thetranslation initiation site of the translational reading frame of thepolypeptide is typically positioned between one and about fiftynucleotides downstream of the promoter. A promoter can, however, bepositioned as much as about 5,000 nucleotides upstream of thetranslation initiation site, or about 2,000 nucleotides upstream of thetranscription start site. A promoter typically comprises at least a core(basal) promoter. A promoter also may include at least one controlelement, such as an enhancer sequence, an upstream element or anupstream activation region (UAR). The choice of promoters to be includeddepends upon several factors, including, but not limited to, efficiency,selectability, inducibility, desired expression level, and cell- ortissue-preferential expression. It is a routine matter for one of skillin the art to modulate the expression of a coding sequence byappropriately selecting and positioning promoters and other regulatoryregions relative to the coding sequence.

The vectors also can include, for example, origins of replication,scaffold attachment regions (SARs), and/or markers. A marker gene canconfer a selectable phenotype, e.g., antibiotic resistance, on a cell.In addition, an expression vector can include a tag sequence designed tofacilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such as greenfluorescent protein (GFP), glutathione S-transferase (GST),polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven,Conn.) sequences typically are expressed as a fusion with the encodedpolypeptide. Such tags can be inserted anywhere within the polypeptide,including at either the carboxyl or amino terminus.

The expression vectors disclosed herein containing the above describedcoding can be used, for example, to transfect or transduce eitherprokaryotic (e.g., bacteria) cells or eukaryotic cells (e.g., yeast,insect, or mammalian) cells. Such cells can then be used, for example,for large or small scale in vitro production of the relevant fusionprotein by methods known in the art. In essence, such methods involveculturing the cells under conditions which maximize production of thefusion protein and isolating the fusion protein from the cells or fromthe culture medium.

Conjugates. In another embodiment, the ligand and immunogen can beobtained separately, either through chemical synthesis or synthesis invivo, purified and then linked non-covalently or covalently. A usefulnon-covalent linkage is a biotin-avidin linkage. The binding of biotinto avidin or streptavidin is essentially irreversible, with a reportedKd of 10⁻¹⁵M. The term “biotin-avidin linkage” as used herein refers toany linkage via biotin or a biotin derivative or biomimic (e.g.,Strep-Tag (EBA, St. Louis, Mo.)) and avidin or an avidin derivative,streptavidin, or biotin-binding fragments or subunits of avidin orstreptavidin.

Thus, the biotinylated ligand can be linked to a biotinylated immunogenvia avidin or streptavidin, or biotin-binding fragments or subunits ofavidin or streptavidin. Methods for forming biotin-avidin linkages arewell known to those in the art. (See for example, Handbook of AffinityChromatography, (Chromatographic Sciences Series, vol. 63) ed. T. Kline,ISBN: 0824789393—Marcel Dekker (1993). Avidin and avidin derivatives areavailable from commercial sources (Pierce Biotechnology, Rockford, Ill.;Invitrogen, Carlsbad, Calif.).

The ligand and the immunogen can also be linked through abiotin-streptavidin linkage that includes an additional biotinylatedimmunoglobulin. For example, a biotinylated ligand can be linked to anavidin molecule that is bound to a biotinylated antibody thatspecifically binds the immunogen.

Avidin and streptavidin have four biotin-binding sites each. By varyingthe relative ratios of the ligand and the immunogens used in theassembly of the targeted immune conjugates, it is possible to generatetargeted immune conjugates with various ratios of linker: immunogen.Thus, biotin-avidin heterocomplexes can be prepared to include 1molecule of the ligand and 3 molecules of the immunogen; 2 moleculeseach of ligand and immunogen; or 3 molecules of ligand and 1 molecule ofimmunogen. Assembly of the biotin-avidin linkages can be performed inany order. The composition of the assembled immune conjugates can bevalidated by SDS-PAGE and western blotting and LC/MS methods.

Alternative docking pairs of molecules with ultra-high affinity (10⁻¹⁰Mor more) may be used in place of biotin-avidin. An example of such apair is vitamin B12 (cyanocobalamin), which is bound by vitaminB12-binding protein with a Kd of 10⁻¹⁰M).

The ligand and the immunogen can also be synthesized as separateentities (by either chemical synthetic or recombinant methods) and thenlinked together by standard chemical methods known in the art. Chemicalcross-linking agents can be homo-bifunctional (the same chemicalreaction takes place at each end of the linker) or hetero-bifunctional(different chemical reactions take place at the ends of the linker). Thechemistries available for such linking reactions include, but are notlimited to, reactivity with sulfhydryl, amino, carboxyl, diol, aldehyde,ketone, or other reactive groups using electrophilic or nucleophilicchemistries, as well as photochemical cross-linkers using alkyl oraromatic azido or carbonyl radicals. An example of a targeted conjugatecoupled via a homobifunctional cross-linking reagent can be a complex ofan anti-band 3 monoclonal antibody as the red blood cell-targetingcomponent and anthrax protective antigen as the immunogen linked bydisuccinimidyl suberate (DSS, Pierce, Rockford, Ill.). An example of atargeted conjugate coupled via a heterobifunctional cross-linkingreagent can be an anti-glycophorin A monoclonal antibody as thetargeting component and a non-toxic fragment of botulinum neurotoxin Aas the immunogen linked by N-succinimidyl3-[2-pyridyldithio]-propionamido (SPDP). In this example, the antibodyis first derivatized at sulfhydryl groups with SPDP's pyridyldithioreactivity, followed by the addition of the toxin, whose amino residuesreact with SPDP's succinimidyl groups.

Examples of chemical cross-linking agents include, without limitation,glutaraldehyde, carbodiimides, bisdiazobenzidine, andN-maleimidobenzoyl-N-hydroxysuccinimide ester. Chemical cross-linkersare widely available from commercial sources (e.g., Pierce Biotechnology(Rockford, Ill.); Invitrogen (Carlsbad, Calif.); Sigma-Aldrich (St.Louis, Mo.); and US Biological (Swampscott, Mass.)).

In another embodiment, the ligand and the immunogen can be connectedthrough a linking polymer. Examples of linking molecules include, butare not limited to linear or branched polymers or co-polymers (e.g.polyalkylene, poly(ethylene-lysine), polymethacrylate, polyamino acids,poly- or oligosaccharides, dendrimers). The ligand and the immunogen canbe attached to the linking molecule or microparticle through anon-covalent high affinity linkage, e.g., streptavidin-biotin highaffinity binding or chemical cross-linking techniques as describedabove.

For example, a polymer-supported targeted immunogen conjugate can beformed using a poly(ethylene-lysine) backbone. Such a linear copolymerbackbone can be synthesized using bis(succinimidyl) poly(ethyleneglycol₂₀₀₀) (Fluka Chemicals) to react with the α and ε amino groups oflysine. The available carboxyl termini of the lysines can be activatedusing (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride)(EDC, Pierce Biotechnology) in preparation for coupling amine-containingcompounds. The total length of the co-polymer can be determined, inpart, by the duration of the polymerization reaction. The targeting andIMG units can be combined in various ratios since there are up to 10positions in a 2000 dalton co-polymer built with, e.g., PEG units of2,000 dalton. Co-polymers using shorter PEG units (e.g., 500 daltons)can also be synthesized.

One example of a polymer-based targeted immunogen conjugate can be theaddition of equimolar amounts of a targeting monoclonal antibody (e.g.anti-CR2) and immunogen (e.g., influenza virus peptide M2e) to produce acomplex with a co-polymeric scaffold studded with targeting antibodymolecules as well as immunogen molecules.

In another embodiment, the ligand and the immunogen can be connectedthrough a microparticle. Examples of linking microparticles include, butare not limited to, micelles, liposomes, fullerenes, nanotubes, or othercolloidal complexes such as lipoproteins. Liposomes and micelles can beprepared by methods described in Lasic DD, 1998, TIBTech 16:307.Fullerenes and nanotubes can be purchased from American Dye Source(www.adsdyes.com). Lipoproteins can be purchased from BiodesignInternational (www.biodesign.com).

The ligand and the immunogen can be attached to the linking molecule ormicroparticle through a non-covalent high affinity linkage, e.g.,avidin-biotin high affinity binding or chemical cross-linking techniquesas described above. Alternatively or in addition, the ligand and/or theimmunogen can be adsorbed or incorporated into a hydrophobicmicroparticle by hydrophobic affinity. A ligand and/or and immunogenwith an available hydrophobic domain can spontaneously associate with ahydrophobic microparticle by hydrophobic partitioning. The hydrophobicdomain on the ligand and/or immunogen can be a polyamino acid stretchcomprised of repeating or mixed hydrophobic amino acids (e.g., poly-Ala,poly-Gly, poly-Leu, poly-Ile, or Ala-Gly-Leu-Ile (SEQ ID NO:5), etc.) ora bilayer-spanning polypeptide from a known trans-membrane protein, suchas membrane IgM), alkyl chains (e.g., fatty acyl), or other hydrophobicstructure (e.g., steroid). Such hydrophobic sequences can be naturallyoccurring sequences within the ligand and/or immunogen. Alternatively,such sequences can be introduced into the native amino acid sequence ofthe ligand or immunogen by standard recombinant DNA technology. Therecombinant protein can be expressed and purified as described above.

The immune conjugates disclosed herein can include one or more of thesame ligands or any combination of different ligands. The immuneconjugate can also include one or more of the same immunogens or anycombination of different immunogens. Thus, the immune conjugates caninclude immunogens that contain multiple copies (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 10, 30, or more) of a single antigen or a single copy ofmultiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more)antigens or multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10,30, or more) of two or more antigens (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 10, 30, or more). The immunogen can contain one or more copies ofone or more peptide epitopes together with one or more copies of any ofthe non-peptide epitopes, e.g. post-transcriptional modifications,carbohydrates, or lipopolysaccharides.

Further, a targeted immune conjugate can include an immunogen thatincludes more than one polypeptide, or any combination of differentpolypeptides. It is noted that each polypeptide in a composition canhave an identical amino acid sequence. In addition, the polypeptides ina composition can contain different amino acid segments, each of whichcan act as a defined immunogenic unit against which an immune responseis desired. Thus, the polypeptides in a composition can containdifferent amino acid segments that correspond to any region from apolypeptide including, without limitation, receptor binding regions,ligand binding regions, enzyme active sites, enzyme cleavage sites ofpolypeptide substrates, antigen-binding regions of antibodies, andepitopes recognized by antibodies. Typically, the administration of apolypeptide results in the formation of antibodies having specificityfor an epitope or combination of epitopes formed by the amino acidsegments within one or more of the polypeptides in the composition.

III. Methods of Use

The immune conjugates disclosed herein are generally useful forgenerating immune responses and as prophylactic vaccines or immuneresponse-stimulating therapeutics. As used herein, “prophylaxis” canmean complete prevention of the symptoms of a disease, a delay in onsetof the symptoms of a disease, or a lessening in the severity ofsubsequently developed disease symptoms. As used herein, “therapy” canmean a complete abolishment of the symptoms of a disease or a decreasein the severity of the symptoms of the disease.

The methods disclosed herein can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys), horses, cattle, pigs,sheep, deer, elk, goats, dogs, cats, mustelids, rabbits, guinea pigs,hamsters, rats, and mice. Thus, they can be used, for example, asvaccines or therapeutic agents against infectious diseases, includingdiseases that can potentially result from bioterrorism attacks. Theimmune conjugates can be used in the preparation of a medicament fortreatment of an infectious disease. Infectious diseases can includediseases caused by any of the pathogens listed herein. Examples include,without limitation, influenza, HIV-AIDS, hepatitis, botulism, plague,smallpox, tularemia, viral hemorrhagic fevers, brucellosis,gastrointestinal disease induced by pathogenic forms of E. coli,Salmonella and Shigella, glanders, melioidosis, psittacosis, Q fever,Staph infection, typhus fever, viral encephalitis, water and foodbornesafety threats, cholera, diphtheria, endocarditis, Legionaire's disease,Listeriosis, periodontal disease, Asperigillosis, Blastomycosis,histoplasmosius, trypanosomiasis, malaria, Giardiasis, Schistosomiasis,toxoplasmosis, smallpox, west Nile virus.

In addition, the immune conjugates can be useful as both prophylacticsand therapeutics for cancer (e.g., any of those recited above). Theimmune conjugates can be employed to stimulate an immune responseagainst cells in a cancer patient or can be administered in cases wherea subject is at relatively high risk for a cancer (e.g., lung cancer ina tobacco smoker or melanoma in a subject with multiple nevi). Moreover,as described above, the immune conjugates can also be useful in therapyor prophylaxis of neurodegenerative diseases. Thus the immune conjugatescan be administered to an individual with Alzheimer's disease or TSE oradministered to an individual who is at risk for developing Alzheimer'sdisease or TSE.

Immune conjugates disclosed herein can also be useful as a contraceptivevaccine, when the immunogen is a germ cell antigen.

The immune conjugates disclosed herein can also be useful asprophylactics and therapeutics against medical conditions that resultfrom exposure to toxins. Such targeted immune conjugates that includenon-toxic variants of toxic substances, e.g., ricin, botulinum toxin,nicotine and other drugs, can be used to stimulate an immune responseagainst the harmful form of the toxin, and thus protect against ormitigate the potential damage the toxin or drug may cause.

The immune conjugates can be administered directly to a mammal. Theimmune conjugates can be used in the preparation of a medicament.Generally, the immune conjugates can be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline). Acomposition can be made by combining any of the immune conjugatesprovided herein with a pharmaceutically acceptable carrier. Suchcarriers can include, without limitation, sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsinclude mineral oil, propylene glycol, polyethylene glycol, vegetableoils, and injectable organic esters, for example. Aqueous carriersinclude, without limitation, water, alcohol, saline, and bufferedsolutions. Preservatives, flavorings, and other additives such as, forexample, antimicrobials, anti-oxidants, chelating agents, inert gases,and the like also may be present. It will be appreciated that anymaterial described herein that is to be administered to a mammal cancontain one or more pharmaceutically acceptable carriers.

Any composition described herein can be administered to any part of thehost's body. A composition can be delivered to, without limitation, thejoints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, orperitoneal cavity of a mammal. In addition, a composition can beadministered by intravenous, intraperitoneal, intramuscular,subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal,intratracheal, intradermal, or transdermal injection, by oral or nasaladministration, by inhalation, or by gradual perfusion over time. In afurther example, an aerosol preparation of a composition can be given toa host by inhalation.

The dosage required depends on the route of administration, the natureof the formulation, the nature of the patient's illness, the subject'ssize, weight, surface area, age, and sex, other drugs beingadministered, and the judgment of the attending physician. Suitabledosages are in the range of 0.01-1,000 μg/kg. Wide variations in theneeded dosage are to be expected in view of the variety of immuneconjugates available and the differing efficiencies of various routes ofadministration. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2- or 3-, 4-,6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of theimmune conjugate in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery.

The duration of treatment with any composition provided herein can beany length of time from as short as one day to as long as the life spanof the host (e.g., many years). For example, an immune conjugate can beadministered once a month for three months or once a year for a periodof ten years. It is also noted that the frequency of treatment can bevariable. For example, an immune conjugate can be administered once (ortwice, three times, etc.) daily, weekly, monthly, or yearly.

Alternatively or in addition the immune conjugates can be administeredalong with an adjuvant. An “adjuvant” is an immunological compound thatcan enhance an immune response against a particular antigen such as apolypeptide. Examples of adjuvants include alum and other aluminum-basedcompounds (e.g., Al₂O₃). Aluminum-based compounds can be obtained fromvarious commercial suppliers. Other adjuvants include immuno-stimulatingcomplexes (ISCOMs) that can contain such components as cholesterol andsaponins; one or more additional immunostimulatory components,including, without limitation, muramyldipeptide (e.g.N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP), monophosphoryl-lipid A(MPL), and formyl-methionine containing tripeptides such asN-formyl-Met-Leu-Phe. Such compounds are commercially available fromSigma Chemical Co. (St. Louis, Mo.), for example. Other adjuvants caninclude CpG oligodeoxynucleotides (Coley Pharmaceuticals), QS21(Cambridge Biotech) and MF59 (Chiron).

The compositions provided herein can contain any ratio of adjuvant toimmune conjugate. The adjuvant:immune conjugate ratio can be 50:50(vol:vol), for example. Alternatively, the adjuvant:immune conjugateratio can be, without limitation, 90:10, 80:20, 70:30, 64:36, 60:40,55:45, 40:60, 30:70, 20:80, or 90:10.

An effective amount of any composition provided herein can beadministered to a host. The term “effective” as used herein refers toany amount that induces a desired immune response while not inducingsignificant toxicity in the host. Such an amount can be determined byassessing a host's immune response after administration of a knownamount of a particular composition. In addition, the level of toxicity,if any, can be determined by assessing a host's clinical symptoms beforeand after administering a known amount of a particular composition. Itis noted that the effective amount of a particular compositionadministered to a host can be adjusted according to a desired outcome aswell as the host's response and level of toxicity. Significant toxicitycan vary for each particular host and depends on multiple factorsincluding, without limitation, the host's disease state, age, andtolerance to pain.

Any method can be used to determine if a particular immune response isinduced. For example, antibody responses against a particular immunogencan be determined using an immunological assay (e.g. ELISA or lymphocyteproliferation assay). In such an assay, the wells of a microtiter platecan be coated with the immunogen and incubated with serum from a mammaltreated with the immune conjugate designed to produce antibodies againstthe corresponding immunogen in that mammal, and the presence or absenceof antibodies against the immunogen can be determined by standardmethods know to those in the art. In addition, clinical methods that canassess the degree of a particular disease state can be used to determineif a desired immune response is induced. For example, in a cancerpatient, a reduction in tumor burden can indicate a desired immuneresponse in a patient treated with a composition designed to stimulatean immune response against a tumor antigen expressed on the patient'stumor.

Alternatively, a polynucleotide containing a nucleic acid sequenceencoding an immune conjugate of interest can be delivered to anappropriate cell of the animal. This can be achieved by, for example,the use of a polymeric, biodegradable microparticle or microcapsuledelivery vehicle, sized to optimize phagocytosis by phagocytic cellssuch as macrophages. For example, PLGA (poly-lacto-co-glycolide)microparticles approximately 1-10 μm in diameter can be used. Thepolynucleotide is encapsulated in these microparticles, which are takenup by macrophages and gradually biodegraded within the cell, therebyreleasing the polynucleotide. Once released, the DNA is expressed withinthe cell. A second type of microparticle is intended not to be taken updirectly by cells, but rather to serve primarily as a slow-releasereservoir of nucleic acid that is taken up by cells only upon releasefrom the micro-particle through biodegradation. These polymericparticles should therefore be large enough to preclude phagocytosis(i.e., larger than 5 μm and preferably larger than 20 μm).

Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors can be incorporated alone intothese delivery vehicles or co-incorporated with tissue-specificantibodies. Alternatively, one can prepare a molecular conjugatecomposed of a plasmid or other vector attached to poly-L-lysine byelectrostatic or covalent forces. Poly-L-lysine binds to a ligand thatcan bind to a receptor on target cells. Delivery of “naked DNA” (i.e.,without a delivery vehicle) to an intramuscular, intradermal, orsubcutaneous site, is another means to achieve in vivo expression.

In the relevant polynucleotides (e.g., expression vectors) the nucleicacid sequence encoding the fusion protein of interest with an initiatormethionine and optionally a targeting sequence is operatively linked toa promoter or enhancer-promoter combination. Promoters and enhancers aredescribed above.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles which are suitable for administration to a human orother mammalian subject, e.g., physiological saline. A therapeuticallyeffective amount is an amount of the polynucleotide which is capable ofproducing a medically desirable result (e.g., a T cell response) in atreated mammal. As is well known in the medical arts, the dosage for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Dosages will vary, but a preferred dosage foradministration of polynucleotide is from approximately 10⁶ to 10¹²copies of the polynucleotide molecule. This dose can be repeatedlyadministered, as needed. Routes of administration can be any of thoselisted above.

The immune conjugates provided herein can be administered in conjunctionwith other therapeutic modalities to an individual in need of therapy.The immune conjugates can be given prior to, simultaneously with orafter treatment with other agents. In the case of infectious disease,the immune conjugates can be administered in conjunction with anyantimicrobial agent, e.g. an antibiotic, e.g. including, withoutlimitation, aminoglycosides, cephalosporins, macrolides, penicillins,peptides, quinolones, sulfonamides, tetracyclines; an antiviral,including without limitation, amantadine, rimantadine, zanamavir andoseltamivir; an anti-fungal, including, without limitation,echinocandin, caspofungin, anidulafungin; or anti-parasitic agent,including, without limitation, chlorquine, mebendazole, andclotrimazole.

The immune conjugates can also be used in conjuction with standardanti-cancer therapies, including, without limitation, chemotherapy,e.g., alkylating agents, anthracyclines, cycloskeletal disruptors,topoisomerase inhibitors, nucleotide analogues, platinum-based agents,retinoids, vinca alkaloids; radiation therapy, hormone ablation andsurgery. The immune conjugates can also be used in conjunction withother therapeutics for neurodegenerative diseases, including donepezil,galantamine, memantine.

In vitro application of the immune conjugates can be useful, forexample, in basic scientific studies of immune mechanisms or forproduction of activated T cells for use in either studies on T cellfunction or, for example, passive immunotherapy.

Articles of Manufacture

Also disclosed are articles of manufacture that can include immuneconjugates as provided herein. Components and methods for producingarticles of manufacture are well known. An article of manufacture caninclude, for example, one or more immune conjugates. In addition, anarticle of manufacture further may include, for example, packagingmaterials, instructions for use, buffers or other control reagents fortreating or monitoring the condition for which prophylaxis or treatmentis required.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1 Assembly of Tripartite Conjugates of the BiotinylatedMonoclonal Antibody Anti-TER-119, Streptavidin and the BiotinylatedPeptide M2E

Conjugates to deliver the immunogen M2e, to the mouse red blood cell(mRBC), were prepared by coupling the peptide with the rat anti-mousemonoclonal antibody anti-TER-119 and using streptavidin (StAv) as thelinker.

Preparation of tripartite immune conjugates. The peptide M2e(SLLTEVETPIRNEWGCRCNDSSD) (SEQ ID NO: 1) was produced by peptidesynthesis (BioWorld, Dublin Ohio) to contain a biotinylated lysineresidue at its carboxy terminus. Anti-TER-119, a rat anti-mouse IgG2b,κmonoclonal antibody, was purchased biotinylated from BD-Pharmingen, aswas the isotype control, biotinylated rat IgG2b,κ. The extent ofbiotinylation of these products was not available from the manufacturer.Streptavidin was purchased from Sigma-Aldrich.

Biotinylated M2e peptide (b-M2e) (SEQ ID NO: 1) was incubated with StAvin phosphate-buffered saline, 10 mM sodium phosphate, 140 mM NaCl, pH7.4 (PBS) at ambient temperature at various molar ratios. After 30 minincubation biotinylated anti-TER-119 antibody (b-Ab) was added and theincubation was continued for an additional 30 min with occasionalagitation. Reaction samples were analyzed by 3 to 8% gradient gelelectrophoresis under native (or non-denaturing) conditions (NDGE,Invitrogen, tris-glycine running buffer) and visualized by Coomassieblue staining. The results showed that mixtures of conjugates ofdifferent apparent molecular weights and stoichiometries were formedcaused by differential cross-linking by StAv between the biotinylatedpeptide and the Ab. The relative amounts of reagents used in thereactions are shown in the grids below the respective NDGE images.

Characterization of tripartite immune conjugates. In the experimentdepicted in FIG. 1 the amounts of StAv and b-Ab were kept constant butthe amount of b-M2e was varied as indicated. Pre-incubation of 100 or200 μmol b-M2e with 20 pmol StAv, which is well in excess of the 80 pmolrequired to saturate the StAv biotin-binding sites, resulted information of StAv(M2c)₄. This conjugate migrated faster than free StAvand similarly to unreacted b-M2e (lanes 10-12 compared to lane 3).

Because the mobility of reagents in NDGE is not solely determined bytheir molecular weight but also by their conformation and charge, StAvmigrated considerably faster than Ab, to a location on the gel that wasslightly above the peptide b-M2e. Addition of b-M2e peptide to StAv,while increasing its MW, caused a shift in the complex's migrationdownward, to the position of unreacted b-M2e (lanes 3 and 6). That thiswas the effect of binding of b-M2e to StAv was demonstrated by thereaction in lane 4, in which excess non-biotinylated peptide M2e did notalter the mobility of StAv). When b-M2e+StAv mixtures containing anexcess of b-M2e were combined with b-Ab there was no shift in themigration of the Ab (lanes 11 and 12, compared to lane 5).

The admixture of 50 pmol or less b-M2e, which was below the theoretical80 pmol needed to saturate 20 pmol StAv, did shift the mobility of theadded b-Ab upward indicating the presence of Ab-StAv_(n), where nrepresents the number of biotin residues per Ab molecule. In addition,the laddering pattern in lanes 7-10, indicated the presence of possiblehigher order complexes such as Ab-StAv-Ab, Ab-StAv2-Ab2,Ab-StAv-Ab-StAv-Ab, etc. Useful tripartite complexes were those that didnot include unreacted Ab; these complexes had a mobility that wasshifted upward from that of the unconjugated antibody, but not to adegree that prevented the complexes from entering the gel, e.g.,

in lanes 9-10, where the ratio of tetravalent StAv:b-M2e wasapproximately 1:3.

Conditions for the formation of the Ab-StAv-M2e tripartite immuneconjugates were analyzed in the experiment shown in FIG. 2. Theconcentration of b-Ab was held constant at 12.5 pmol, and differentamounts of StAv-b-M2e conjugates pre-formed at a 1:3 ratio were added.Complexes of various stoichiometries were seen in all combinations, withthe higher order complexes seen where StAv was in excess over b-Ab(lanes 6-9). FIG. 2 also showed that in the 1:3 StAv:b-M2e mixturesthere were some StAv molecules that had a minimum of 2 freebiotin-binding sites which were responsible for cross-linking b-Abmolecules (lanes 5-9). This experiment also indicated that the antibodymolecules contained more than one biotin residue. If b-Ab weremono-biotinylated, the reaction with free StAv would lead to apreponderance of Ab tetramers, with lesser amounts of trimers, dimmersand monomers. Lane 3 shows that this is not the case. Instead there weremore types of complexes without apparent preponderance of tetramers.

One objective of these experiments was to define conditions thatproduced tripartite conjugates with a maximum number of M2e peptides perAb molecule. The stoichiometric ratios of Ab:StAv:M2e in the experimentsdescribed above are listed in Table 1.

TABLE 1 Molar ratios of the components in tripartite conjugate reactionsdepicted in FIGS. 1 and 2 (normalized to the concentration of Ab). FIG.1 FIG. 2 lane 8 9 10 11 12 5 6 7 8 9 Ab 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.01.0 1.0 StAv 1.3 1.3 1.3 1.3 1.3 0.8 1.6 2.0 2.4 3.2 M2e 0.7 1.7 3.3 6.713.3 2.4 5.4 6.0 7.2 9.6

The conditions in which most of the Ab was shifted by the reaction, butthe complexes formed were not too large to diffuse into the gel arefound in FIG. 1, lanes 9 and 10 and FIG. 2, lanes 6, 7 and 8. The immuneconjugates that included the highest proportion of peptides per Ab (6 or7.2) were those shown in lane 7 or 8 of FIG. 2.

Example 2 Analysis of Anti M2e Activity in Mice Challenged with theImmune Conjugate Biotinylated Rat Anti-Mouse TER-119 Mab−Streptavidin-Biotinylated M2e Peptide

Mice are challenged intraperitoneally with an immune conjugate ofbiotinylated rat anti-mouse TER-119 Mab−streptavidin-biotinylated M2epeptide ((SEQ ID NO: 1)) and their serum antibody response to M2e (SEQID NO: 1) is assayed.

M2e peptide (SEQ ID NO: 1) with a C-terminal addition oflysyl-ε-N-biotin or PGGG (SEQ ID NO:6) is synthesized by solid phasepeptide synthesis (R10-World). This peptide is coupled to streptavidin(StAv) (Sigma-Aldrich) at a peptide:StAv molar ratio of 3:1 by mixingwith b-Ab, as described in Example 1. Identical tripartite conjugate isprepared using biotinylated rat IgG (BD Pharmingen) as a control. Toprepare TER-119-StAv and rat IgG-StAv conjugates the StAv is firstreacted at a biotin:StAv molar ratio of 3:1 to occupy 3 of the 4biotin-binding sites (BBSs) on each StAv molecule. In groups receivinguncoupled StAv, it is first reacted with a minimum of 4 molarequivalents of biotin to saturate all the BBSs. M2e peptide conjugatedto keyhole limpet hemocyanin (KLH) is produced by Bio-World.

Groups of 5 mice each (Balb/C, female, 6-8 weeks old) are injectedintravenously (i.v.) in the tail vein with a range of free andconjugated peptide doses (0.01 to 10 μg equivalent of peptide in 50 μlPBS). Since StAv and TER-119 (a rat IgG) are also immunogenic in mice,Ab titers against them alone or in RBC-targeted conjugates are alsoevaluated. Groups 1-6 are the minimum set to analyze the relativeanti-M2e responses. Groups 7-15 include additional controls to furtheranalyze the anti-StAv and anti-rat IgG responses, as well as the effectof simply mixing the various components, instead of coupling them intotargeted conjugates. The experimental design is depicted in the tablebelow:

TABLE 2 Experimental design: Example 2. Group number Ligand LinkerImmunogen 1 Biotinylated Anti- streptavidin Biotinylated M2e TER119 2Biotinylated Rat streptavidin Biotinylated M2e IgG 3 — streptavidinBiotinylated M2e 4 KLH Chemical conjugate M2e 5 — — M2e 6 (PBS — — —injected only) 7 Biotinylated Anti- streptavidin — TER119 8 BiotinylatedRat streptavidin — IgG 9 Biotinylated Anti- streptavidin M2e TER119 10Biotinylated Rat streptavidin M2e IgG 11 — streptavidin M2e 12Biotinylated Anti- — M2e TER119 13 Biotinylated Rat — M2e IgG 14Biotinylated Anti- — — TER119 15 Biotinylated Rat — — IgG

The mice are boosted with a repeat i.v. injection after 2 and againafter 4 weeks. Blood samples (˜100 μl) are collected retro-orbitallyprior to and every week after the primary injection for 5 weeks. Bloodsamples are chilled on ice and allowed to clot overnight. The tubes arecentrifuged and serum is collected and stored at −20° C.

Mouse IgG titers against all 3 antigens (Ags), M2e, StAv and rat IgG,are determined by ELISA. ELISA plates (Immulon, VWR) are coated with 100μl Ag at 10 μg/ml in sodium carbonate (Na₂CO₃) buffer, 50 mM, pH 9.6overnight at 4° C. Plates are washed and blocked with 200 μl/well of 3%BSA in PBS containing 0.05% Tween-20 (PBST) for 1.5 hours at roomtemperature (RT). Wash 3× with 200 μl/well of PBST. Dispense 100 μl/wellof individual mouse sera at 1:50 dilution in PBST in duplicates andprepare serial dilutions in 1:3 X increments. Allow mouse antisera tobind for 1.5 hours at RT and wash the plates as above. Add 100 μl/wellgoat anti-mouse IgG conjugated with horseradish peroxidase (diluted pervendor instructions, Sigma), incubate for 1 hour at RT, and wash asabove. Add 100 μl/well substrate (SureBlue, KPL) and read absorbance at405 nm. Positive control Abs are: Mouse Mab 14C2 which recognizes theN-terminal ectodomain epitope of M2 (Abeam), mouse Mab anti-StAv (Abeam)and mouse polyclonal anti-rat IgG (H & L chains) (Invitrogen).

Example 3 Analysis of Anti M2e Activity in Mice Challenged with theImmune Conjugate Biotinylated Rat Anti-Mouse TER-119Mab−Streptavidin-Biotinylated M2e Peptide (SEQ ID NO: 1): Comparison ofDifferent Routes of Administration

The effect of the route of administration on the serum antibody responseto M2e peptide in mice challenged with an immune conjugate ofbiotinylated rat anti-mouse TER-119 Mab+streptavidin-biotinylated M2epeptide is evaluated by comparing serum titers of anti-M2e antibodies inmice immunized intraperitoneally, intravenously, subcutaneously andintramuscularly. The full Mab-StAv-IMG conjugate is compared to thenon-targeted StAv-IMG partial complex, as well as to PBS vehicle. Thedose will be 1.0 μg equivalent of M2e peptide (SEQ ID NO: 1).Immunizations and ELISA analysis are performed as described in Example2.

Example 4 Analysis of Anti-M2e Fecal IgG and IgA Activity in MiceChallenged with the Immune Conjugate Biotinylated Rat Anti-Mouse TER-119Mab−Streptavidin-Biotinylated M2e Peptide (SEQ ID NO: 1)

Mice are dosed with TER-119−StAv−M2e conjugate and controls as depictedin Table 2 and following the dosing schedule in Example 2:

TABLE 3 Experimental design: Example 4. Group number Ligand LinkerImmunogen 1 Biotinylated Anti- streptavidin Biotinylated M2e TER119 2Biotinylated Rat streptavidin Biotinylated M2e IgG 3 — streptavidinBiotinylated M2e 4 KLH Chemical conjugate M2e 5 — — M2e 6 (PBS — — —injected only)Feces are collected at the times described in Example 2. Soybean trypsininhibitor (0.1 mg/ml in PBS) is added to every 0.1 g of feces, which arevortexed in a mini-beadbeater (Biospec Products) for 10 sec at 2500 rpm,and debris is removed by centrifugation at 9000×g at 4° C. for 15 min.The supernatant is assayed for anti-M2e IgG titer in the ELISA set-updescribed in Example 2, but also for IgA by adding anti-IgA 2^(nd)antibody (goat anti-mouse IgA conjugated with horseradish peroxidase(Sigma-Aldrich).

Example 5 Analysis of Anti-M2e Activity in Mice Challenged with theImmune Conjugate Biotinylated Rat Anti-Mouse CD21/CD35Mab−Streptavidin-Biotinylated M2e Peptide (SEQ ID No: 1)

Mice are injected with an immune conjugate of biotinylated ratanti-mouse CD21/CD35 Mab+streptavidin-biotinylated M2e peptide(influenza) and their serum antibody response to M2e is assayed. Theanti-CD21/CD35 antibody is purchased from eBioscience. The conjugate isprepared according to the method described in Example 2. Groups of 5mice each (Balb/C, female, 6-8 weeks old) are injected intravenously(i.v.) with a range of free and conjugated peptide doses (0.01 to 10 μgequivalent of peptide in 50 μl PBS) according to the experimental designin shown in Table 4 below.

TABLE 4 Experimental design: Example 5. Group number Ligand LinkerImmunogen 1 Biotinylated Anti- streptavidin Biotinylated M2e CD21/CD35 2Biotinylated Rat streptavidin Biotinylated M2e IgG 3 — streptavidinBiotinylated M2e 4 KLH Chemical conjugate M2e 5 — — M2e 6 (PBS — — —injected only) 7 Biotinylated Anti- streptavidin — CD21/CD35 8Biotinylated Rat streptavidin — IgG 9 Biotinylated Anti- streptavidinM2e CD21/CD35 10 Biotinylated Rat streptavidin M2e IgG 11 — streptavidinM2e 12 Biotinylated Anti- — M2e CD21/CD35 13 Biotinylated Rat — M2e IgG14 Biotinylated Anti- — — CD21/CD35 15 Biotinylated Rat — — IgG

Immunizations and ELISA analysis will be performed as described inExample 2.

Example 6 Analysis of Anti-M2C Activity in Mice Challenged with theImmune Conjugate Biotinylated Plasmodium falciparum EBA-175 Peptide1085-1096 (SEQ ID No: 7) (pEBA)+Streptavidin-Biotinylated M2e Peptide

Since Plasmodium falciparum EBA-175 peptide 1085-1096 binds human, butnot mouse RBCs, the mouse experiments are performed in humanGphA-transgenic mice (see Auffrey, et al., 2001, Blood 97:2872-2878.Glycophorin A dimerization and band 3 interaction during erythroidmembrane biogenesis: in vivo studies in human glycophorin A transgenicmice). Alternately, the corresponding binding protein derived from otherspecies of Plasmodium, e.g., P. bergheii or P. yoelii yoelii, which bindmouse RBCs is used (referred to as pEBA_(m)). Complexes are assembledand immunizations and ELISA analysis are performed as described inExample 2.

Example 7 Analysis of Anti-M2e Activity in Mice Challenged with theSynthetic Peptide pEBA-Glycine-Glycine-Glycine-M2e Peptide

Mice are injected intravenously with a synthetic peptide of the sequencepEBA_(m)-175 SEQ ID NO: 7 linked via three glycine residues to the M2epeptide (SEQ ID NO: 1) described in Example 1 above and according to thescheme depicted in Table 5. Each mouse receives a range of free andconjugated peptide doses (0.01 to 10 μg equivalent of M2e peptide in 50μl PBS) intravenously and blood samples are collected and analyzedaccording to the schedule in Example 2.

TABLE 5 Experimental Design: Example 7. Group number Ligand Linkerimmunogen 1 pEBA_(m) Gly-Gly-Gly M2e 2 pEBA_(m) (scrambled) Gly-Gly-GlyM2e 3 KLH Chemical conjugate M2e 4 — — M2e 5 (PBS — — — injected only)

Example 8 Analysis of Anti-HBV Peptide (preS1 Amino Acids 34-59 (SEQ IDNO:2), or pHBV) Activity in Mice Challenged with the Immune ConjugateBiotinylated Rat Anti-Mouse TER-119 Mab+Streptavidin-Biotinylated pHBV

Mice are challenged with immune conjugates in which the immunogen is anHBV (hepatitis B virus) peptide preS1 amino acids 34-59 (Hu W G, et al.2005. World J Gastroenterol. 11:2088-2094. Identification of theimmunogenic domains in HBsAg preS1 region using overlapping preS1fragment fusion proteins) or pHBV coupled to biotinylated TER-119 Mab.Experimental groups, immunizations and ELISA analysis are as describedin Example 2.

Example 9 Analysis of Anti-M2e Activity in Mice Challenged with theImmune Conjugate Biotinylated Mouse Anti-Mouse Band 3Mab−Streptavidin-Biotinylated M2e Peptide (SEQ ID NO:1)

Mouse anti-mouse band 3 Mabs have been produced in several labs from NZBmice. The immune conjugate, biotinylated mouse anti-mouse band 3Mab−streptavidin-biotinylated M2e peptide, is produced according to themethod in Example 1 with an IgG anti-mouse band-3 Mabs (e.g., 34-3C orclass-switched 4C8 (Fossati-Jimack, 2002, J Autoimmun 18:17-25.Selective increase of autoimmune epitope expression on aged erythrocytesin mice: implications in anti-erythrocyte autoimmune responses.)), andis evaluated according to the protocol in Example 2.

Example 10 Optimization of the Degree of Biotinylation of the TargetingMabs

Establish the optimal number of StAv-IMG units to couple to thebiotinylated Mab. Maximize IMG load while retaining full target bindingcapacity of the Mab.

The FluoReporter Biotin Quantitation Kit (Invitrogen) is used forestimating the molar ratio of biotin:protein. Mab anti-TER-119 isbiotinylated using amino-reactive (Pierce EZ-link NHS biotin) andcarbohydrate-reactive (Pierce EZ-link hydrazide-biotin) reagents atvarious levels by adding a range of biotinylation molar excess factors(e.g., 3×, 5×, 10×, 50×, 250×). Add a 5× molar excess StAv per mole ofbiotinylated Mab to saturate all biotinylated sites. Test functionalintegrity of the biotinylated Mab preparations by binding to mouse RBCs.Use goat anti-rat IgG conjugated with fluorescence isothiocyanate (FITC)to detect RBC-bound TER-119 using flow cytometry. Select the conditionthat results in the highest degree of biotinylation of the TER-119 Mabwhile retaining maximum RBC-binding. Use un-biotinylated TER-119 Mab andgoat anti-rat IgG-FITC as the reference.

Example 11 Determination of the Optimal Stoichiometry of Mab−StAv-IMG

The effect of loading 1, 2 or 3 IMGs per StAv and the effect of binding1 or more of these StAv-IMG conjugates per targeting Mab is compared.The optimal construct is identified based on the elicited response invivo (see Example 2). Prepare anti-TER-119+StAv+M2e conjugates atvarious ratios (e.g., 1:1:1, 1:1:3, 1:2:2, 1:2:6, 1:3:3, 1:3:9) andadminister them to mice as in Example 2 (always administer 1.0 μgequivalent of M2e). Compare the resulting anti-M2e titers.

Example 12 Comparison of Monovalent, Divalent and TrivalentPeptide-Mediated Targeting of IMG to RBC

Biotinylated pEBA_(m) (SEQ ID NO:7) (see Example 7) is used as thetargeting ligand and biotinylated M2e is used as the IMG. The followingconstructs are prepared: 1pEBA_(m):1 StAv:3M2e, 2pEBA_(m):1 StAv:2M2eand 3pEBA_(m):1StAv: 1M2e. Inject mice with each of the constructscontaining equal equivalents of M2e (1.0 μg). Evaluate the elicitedimmune response according to the methods in Example 2.

Example 13 Evaluation of the Efficacy of Immunizing with a Construct inwhich the Targeting Component and the IMG are Covalently Bound to aCommon Polymer Backbone

Synthesize each component peptide (e.g., pEBA_(m) (SEQ ID NO:7) and M2e(SEQ ID NO: 1)) with an added Lys whose α-carboxyl group is joined tothe peptide's C-terminus, leaving the α and ε amino groups of Lys free.Co-polymerize various ratios of the components with bis-succinimidylpoly(ethylene glycol), or BS-PEG (mean molecular weight 2,000 da). Thefree a and a amino groups of Lys are linked to the reactive PEG units tocreate a co-polymer that has the pattern PEG-P-PEG-P-PEG-P-PEG . . . ,where P is either peptide pEBA_(m) or M2e in a random distribution. Theratio of pEBA_(m) to M2e is a function of the ratio of added peptides tothe polymerization reaction. Prepare co-polymer backbone targeted IMGconstructs in which the Mab:IMG ratios are 0:10 (un-targeted multivalentIMG control), 1:9, 3:7, 5:5, 7:3 and 9:1. Evaluate their efficacy byinjecting 1.0 μg equivalents of M2e as described in Example 2.

Example 14 Preparing Targeted Constructs Using a MicroparticulateLinking Component

Liposomes containing a biotinylated lipid in the bilayer (e.g.,phosphatidyl ethanolamine reacted with succinimidyl-biotin) areprepared. The exposed biotin groups are used to bind StAv and then theStAv's biotin-binding sites are loaded with biotinylated targeting andIMG components at various ratios. The comparatively large liposomalsurface permits adding many more units of each component without causingsteric hindrance. In order to prevent StAv from cross-linking theexposed biotin groups on the liposomal surface, pre-load 3 moleequivalents of either targeting or IMG units (or combinations of these)per mole of StAv, and then add this conjugate to the liposomes.Administer and analyze as in Example 2.

Example 15 Preparing Targeted Constructs Using a MicroparticulateLinking Component and a Mixture of IMG's

Biotinylate a mixture of IMGs from a relevant source (e.g., the multiplestrain variants of a multivalent pneumococcal polysaccharide vaccine orthe various proteins or peptides from a viral vaccine) using a suitablereactive biotin reagent (in the case of oligosaccharides use abiotinylation reagent with a hydrazide reactive terminus (EZ-linkhydrazide biotin, Pierce), and in the case of peptidic preparations useone with a terminus containing either a succinimidyl group for reactionwith amines, a maleimidyl group for reaction with sulfhydryls, or anamine terminus reactive with carboxyls in the presence ofethylenediamine carbodiimide (EDC)). Add such a mixture of biotinylatedIMGs to a targeting conjugate consisting of StAv bound to a biotinylatedMab, a biotinylated targeting peptide, such that the StAvs haveremaining unoccupied biotin-binding sites. These are produced byreacting a large molar excess of StAv with the biotinylated targetingcomponent. The biotinylated IMG mixture can also be reacted withliposomes bearing surface StAv molecules bound to lipid-bound biotin(e.g., biotinylated phosphatidyl ethanolamine), with sufficientunoccupied biotin-binding sites remaining on the bound StAv molecules.Administer and analyze as in Example 2.

Example 16 Using Anti-Band 3 Mab for Targeting IMGs to Senescent RBCs

Approximately 1% of RBCs are senescent and destined for removal from thecirculation by phagocytosis in the RES. Their clearance is mediated bypre-existing natural IgG Abs (Nabs). These Nabs have low affinity forband 3. They can only bind their target Ag on the RBC firmly if the band3 molecules are clustered in the RBC membrane, through augmentedavidity. Band 3 clustering occurs as a result of cumulative oxidativedamage in the course of the life of the cells. Use a slightly higheraffinity anti-band 3 Mab, sufficiently high to out-compete the Nabs, butnot so high as to bind all RBCs. The Mab can be a humanized IgG, fortargeting IMGs to senescent RBCs.

To develop a human anti-band 3 Mab, immunize a mouse with human band 3protein or with human red blood cells (which contain a senescentsubpopulation) and fuse the spleen cells with a myeloma fusion partner(e.g., SP2/0). Plate fused cells at 100,000 cells/well in hybridomaselection medium (HAT). After 3 weeks screen supernatants of wells withhybridoma clones by ELISA on wells coated with band 3 proteinextracellular domain. Subclone hybridomas that react specifically withhuman band 3 until the cultures are monoclonal. The resulting murine Mabcan be “humanized” by methods familiar to those skilled in the art(e.g., Recombinant Antibodies (1999), Breitling, F. and Dübel, S.(eds.), John Wiley & Sons). Alternately, human anti-band 3 Mabs can beproduced by fusion of normal human donor B cells, among which are foundthe cells producing anti-band 3 Nabs. Enrich B cells (the CD19+ or CD20+subpopulation) from the peripheral blood mononuclear fraction byimmunomagnetic purification (StemCell Sciences). Fuse the B cells byelectrofusion (CytoPulse) to a suitable fusion partner myeloma orheteromyeloma cell line (e.g., K6H6/A5 or A6 (ATCC) or SP2-IL6-TERT(Dessain S K, et al., 2004, J Immunol Methods 291:109-122. Highefficiency creation of human monoclonal antibody-producing hybridomas.))to form hybridomas. Plate fused cells at 20,000 cells/well in hybridomaselection medium (HAT). After 3 weeks screen supernatants of wells withhybridoma clones by ELISA on wells coated with band-3 proteinextracellular domain. Subclone band 3-specific IgG hybridomas. Purifythe antibodies and use them to prepare an immune conjugate as describedin Example 1. Use biotinylated influenza M2e peptide as the IMG andcouple to the Mab with a StAv bridge. Assay in mice according to themethod in Example 2.

1. An immune conjugate comprising: (a) a ligand that binds specificallyto a cell surface molecule on a circulating non-lymphoid cell of amammal, wherein said molecule comprises glycophorin A, wherein theligand is an antibody; and (b) an immunogen coupled to said ligand,wherein the immunogen comprises an M2e peptide, wherein said immuneconjugate, when administered to an individual, induces or enhances animmune response against said immunogen.
 2. The immune conjugate of claim1, wherein the ligand is biotinylated, glycosylated, acetylated,alkylated, isoprenylated, lipoylated, or phosphorylated.
 3. The immuneconjugate of claim 1, wherein the cell-surface molecule is on a redblood cell.
 4. The immune conjugate of claim 1, wherein the circulatingnon-lymphoid cell is a red blood cell.
 5. The immune conjugate of claim1, wherein the mammal is human.
 6. The immune conjugate of claim 1,wherein the M2e peptide is SEQ ID NO:
 1. 7. The immune conjugate ofclaim 1, wherein the immunogen is biotinylated, glycosylated,acetylated, alkylated, isoprenylated, lipoylated, or phosphorylated. 8.The immune conjugate of claim 1, wherein the immunogen is coupled to theligand via a covalent bond.
 9. The immune conjugate of claim 1, whereinthe immunogen is coupled to the ligand via a peptide bond, wherein theimmune conjugate is a fusion protein.
 10. A nucleic acid sequenceencoding the fusion protein of claim
 9. 11. An expression vectorcomprising the nucleic acid sequence of claim
 10. 12. A host-cellcomprising the expression vector of claim
 11. 13. The immune conjugateof claim 1, wherein the immunogen is coupled to the ligand via anon-covalent bond.
 14. The immune conjugate of claim 13, wherein thenon-covalent bond is a biotin-avidin linkage.
 15. The immune conjugateof claim 1, wherein the immune conjugate comprises two or more of saidligands.
 16. The immune conjugate of claim 1, wherein the immuneconjugate comprises two or more of said immunogens.
 17. A compositioncomprising the immune conjugate of claim 1 and an adjuvant.
 18. A methodof inducing or enhancing an immune response against influenza A in amammalian subject, the method comprising administering a therapeuticallyeffective amount of the immune conjugate of claim 1, wherein an immuneresponse to influenza A is induced in the mammalian subject.
 19. Themethod of treatment of claim 18, wherein the mammalian subject isidentified as suffering from or being at risk for an influenza Ainfection.
 20. An article of manufacture comprising a measured amount ofthe immune conjugate of claim 1 and one or more items selected from thegroup consisting of packaging material, a package insert comprisinginstructions for use, a sterile fluid, and a sterile container.
 21. Animmune conjugate comprising: (a) a ligand that binds specifically to acell surface molecule on a circulating non-lymphoid cell of a mammal,wherein said molecule comprises glycophorin A, wherein the ligand is anantibody; and (b) an immunogen coupled to said ligand, wherein theimmunogen comprises an M2e peptide.
 22. The immune conjugate of claim 1,wherein the M2e peptide is SEQ ID NO:
 3. 23. The immune conjugate ofclaim 1, wherein the M2e peptide is SEQ ID NO: 4.